Combination therapy involving antibodies against claudin 18.2 for treatment of cancer

ABSTRACT

The present invention provides a combination therapy for effectively treating and/or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, and cancer of the gallbladder and metastases thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/231,185, which was filed on Aug. 8, 2016, which is continuation ofU.S. patent application Ser. No. 14/401,557, which was filed on Nov. 17,2014, which issued as U.S. Pat. No. 9,433,675, which is a National StageEntry of PCT/EP2013/001503, which was filed on May 21, 2013 and claimedpriority to PCT/EP2012/002211, which was filed on May 23, 2012. Theentire teachings of the above-referenced Applications are incorporatedherein by reference.

BACKGROUND

Cancers of the stomach and the esophagus (gastroesophageal; GE) areamong the malignancies with the highest unmet medical need. Gastriccancer is the second leading cause of cancer death worldwide. Theincidence of esophageal cancer has increased in recent decades,coinciding with a shift in histological type and primary tumor location.Adenocarcinoma of the esophagus is now more prevalent than squamous cellcarcinoma in the United States and Western Europe, with most tumorslocated in the distal esophagus. The overall five-year survival rate forGE cancer is 20-25%, despite the aggressiveness of established standardtreatment associated with substantial side effects.

The majority of patients presents with locally advanced or metastaticdisease and have to be subjected to first-line chemotherapy. Treatmentregimens are based on a backbone of platinum and fluoropyrimidinederivatives mostly combined with a third compound (e.g. taxane oranthracyclines). Still, median progression free survival of 5 to 7months and median overall survival of 9 to 11 months are the best thatcan be expected.

The lack of a major benefit from the various newer generationcombination chemotherapy regimens for these cancers has stimulatedresearch into the use of targeted agents. Recently, forHer2/neu-positive gastroesophageal cancers Trastuzumab has beenapproved. However, as only ˜20% of patients express the target and areeligible for this treatment, the medical need is still high.

The tight junction molecule Claudin 18 splice variant 2 (Claudin 18.2(CLDN18.2)) is a member of the claudin family of tight junctionproteins. CLDN18.2 is a 27.8 kDa transmembrane protein comprising fourmembrane spanning domains with two small extracellular loops.

In normal tissues there is no detectable expression of CLDN18.2 byRT-PCR with exception of stomach. Immunohistochemistry with CLDN18.2specific antibodies reveals stomach as the only positive tissue.

CLDN18.2 is a highly selective gastric lineage antigen expressedexclusively on short-lived differentiated gastric epithelial cells.CLDN18.2 is maintained in the course of malignant transformation andthus frequently displayed on the surface of human gastric cancer cells.Moreover, this pan-tumoral antigen is ectopically activated atsignificant levels in esophageal, pancreatic and lung adenocarcinomas.The CLDN18.2 protein is also localized in lymph node metastases ofgastric cancer adenocarcinomas and in distant metastases especially intothe ovary (so-called Krukenberg tumors).

The chimeric IgG1 antibody IMAB362 which is directed against CLDN18.2has been developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes thefirst extracellular domain (ECD1) of CLDN18.2 with high affinity andspecificity. IMAB362 does not bind to any other claudin family memberincluding the closely related splice variant 1 of Claudin 18 (CLDN18.1).IMAB362 shows precise tumor cell specificity and bundles fourindependent highly potent mechanisms of action. Upon target bindingIMAB362 mediates cell killing by ADCC, CDC and induction of apoptosisinduced by cross linking of the target at the tumor cell surface anddirect inhibition of proliferation. Thus, IMAB362 lyses efficientlyCLDN18.2-positive cells, including human gastric cancer cell lines invitro and in vivo. Mice bearing CLDN18.2-positive cancer cell lines havea survival benefit and up to 40% of mice show regression of their tumorwhen treated with IMAB362.

The toxicity and PK/TK profile of IMAB362 has been thoroughly examinedin mice and cynomolgus monkeys including dose range finding studies,28-day repeated dose toxicity studies in cynomolgus and a 3-monthrepeated dose toxicity study in mice. In both mice (longest treatmentduration weekly administration for 3 months, highest dose levels 400mg/kg) and cynomolgus monkeys (up to 5 weekly applications of up to 100mg/kg) repeated doses of IMAB362 i.v. are well tolerated. No signs ofsystemic or local toxicity are induced. Specifically, no gastrictoxicity has been observed in any toxicity study. IMAB362 does notinduce immune activation and cytokine release. No adverse effects onmale or female reproductive organs were recorded. IMAB362 does not bindto tissues lacking the target. Biodistribution studies in mice indicatethat the reason for lack of gastric toxicity is most likelycompartimentalization of tight junctions at the luminal site in healthygastric epithelia, which appears to impair accessibility of the IMAB362epitope profoundly. This compartimentalization is lost upon malignanttransformation rendering the epitope drugable by IMAB362.

IMAB362 is in early clinical testing. A phase I clinical study has beenconducted in human. 5 dose cohorts (33 mg/m², 100 mg/m², 300 mg/m², 600mg/m², 1000 mg/m²) of 3 patients each have received a single intravenousadministration of IMAB362 and have been observed for 28 days. IMAB362was very well tolerated, with no relevant safety observation in thepatients. In one patient all measured tumor markers decreasedsignificantly within 4 weeks after treatment. In an ongoing phase IIaclinical study IMAB362 is given repetitively.

The data presented herein indicate that bisphosphonates such aszoledronic acid (ZA), in particular when administered in conjunctionwith recombinant interleukin-2 (IL-2), augment the activity of ananti-CLDN18.2 antibody such as IMAB362. The underlying mechanism isactivation and expansion of a highly cytotoxic immune cell population(γ9δ2 T cells).

Furthermore, we present data demonstrating that chemotherapeutic agentscan stabilize or increase expression of CLDN18.2 on the surface ofcancer cells resulting in an enhanced drugability of CLDN18.2 by ananti-CLDN18.2 antibody such as IMAB362. A synergistic effect of ananti-CLDN18.2 antibody such as IMAB362 with particular chemotherapeuticregimens, in particular chemotherapeutic regimens used for gastriccancer treatment or treatment of human solid cancers was observed. Humancancer cells pre-treated with chemotherapy are more susceptible toantibody-induced target-specific killing. In mouse tumor models, tumorcontrol with an anti-CLDN18.2 antibody plus chemotherapy is superior tothat with an anti-CLDN18.2 antibody as single agent.

SUMMARY OF THE INVENTION

The present invention generally provides a combination therapy foreffectively treating and/or preventing diseases associated with cellsexpressing CLDN18.2, including cancer diseases such as gastric cancer,esophageal cancer, pancreatic cancer, lung cancer such as non small celllung cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer,head-neck cancer, and cancer of the gallbladder and metastases thereof,in particular gastric cancer metastasis such as Krukenberg tumors,peritoneal metastasis and lymph node metastasis. Particularly preferredcancer diseases are adenocarcinomas of the stomach, the esophagus, thepancreatic duct, the bile ducts, the lung and the ovary.

In one aspect, the present invention provides a method of treating orpreventing a cancer disease comprising administering to a patient anantibody having the ability of binding to CLDN18.2 in combination withan agent stimulating γδ T cells. The agent stimulating γδ T cells may beadministered prior to, simultaneously with or following administrationof the antibody having the ability of binding to CLDN18.2, or acombination thereof.

In one embodiment, the γδ T cells are Vγ9Vδ2 T cells. In one embodiment,the agent stimulating γδ T cells is a bisphosphonate such as anitrogen-containing bisphosphonate (aminobisphosphonate). In oneembodiment, the agent stimulating γδ T cells is selected from the groupconsisting of zoledronic acid, clodronic acid, ibandronic acid,pamidronic acid, risedronic acid, minodronic acid, olpadronic acid,alendronic acid, incadronic acid and salts thereof. In one embodiment,the agent stimulating γδ T cells is administered in combination withinterleukin-2.

In one embodiment, the method of the invention further comprisesadministering an agent stabilizing or increasing expression of CLDN18.2.Expression of CLDN18.2 is preferably at the cell surface of a cancercell.

The agent stabilizing or increasing expression of CLDN18.2 may be acytotoxic and/or cytostatic agent. In one embodiment, the agentstabilizing or increasing expression of CLDN18.2 comprises an agentwhich induces a cell cycle arrest or an accumulation of cells in one ormore phases of the cell cycle, preferably in one or more phases of thecell cycle other than the G1-phase. The agent stabilizing or increasingexpression of CLDN18.2 may comprise an agent selected from the groupconsisting of anthracyclines, platinum compounds, nucleoside analogs,taxanes, and camptothecin analogs, or prodrugs thereof, and combinationsthereof. The agent stabilizing or increasing expression of CLDN18.2 maycomprise an agent selected from the group consisting of epirubicin,oxaliplatin, cisplatin, 5-fluorouracil or prodrugs thereof such ascapecitabine, docetaxel, irinotecan, and combinations thereof. The agentstabilizing or increasing expression of CLDN18.2 may comprise acombination of oxaliplatin and 5-fluorouracil or prodrugs thereof, acombination of cisplatin and 5-fluorouracil or prodrugs thereof, acombination of at least one anthracycline and oxaliplatin, a combinationof at least one anthracycline and cisplatin, a combination of at leastone anthracycline and 5-fluorouracil or prodrugs thereof, a combinationof at least one taxane and oxaliplatin, a combination of at least onetaxane and cisplatin, a combination of at least one taxane and5-fluorouracil or prodrugs thereof, or a combination of at least onecamptothecin analog and 5-fluorouracil or prodrugs thereof. The agentstabilizing or increasing expression of CLDN18.2 may be an agentinducing immunogenic cell death. The agent inducing immunogenic celldeath may comprise an agent selected from the group consisting ofanthracyclines, oxaliplatin and combinations thereof. The agentstabilizing or increasing expression of CLDN18.2 may comprise acombination of epirubicin and oxaliplatin. In one embodiment, the methodof the invention comprises administering at least one anthracycline, atleast one platinum compound and at least one of 5-fluorouracil andprodrugs thereof. The anthracycline may be selected from the groupconsisting of epirubicin, doxorubicin, daunorubicin, idarubicin andvalrubicin. Preferably, the anthracycline is epirubicin. The platinumcompound may selected from the group consisting of oxaliplatin andcisplatin. The nucleoside analog may be selected from the groupconsisting of 5-fluorouracil and prodrugs thereof. The taxane may beselected from the group consisting of docetaxel and paclitaxel. Thecamptothecin analog may be selected from the group consisting ofirinotecan and topotecan. In one embodiment, the method of the inventioncomprises administering (i) epirubicin, oxaliplatin and 5-fluorouracil,(ii) epirubicin, oxaliplatin and capecitabine, (iii) epirubicin,cisplatin and 5-fluorouracil, (iv) epirubicin, cisplatin andcapecitabine, or (v) folinic acid, oxaliplatin and 5-fluorouracil.

The method of the invention may further comprise administering at leastone further chemotherapeutic agent which may be a cytotoxic agent.

The antibody having the ability of binding to CLDN18.2 may bind tonative epitopes of CLDN18.2 present on the surface of living cells. Inone embodiment, the antibody having the ability of binding to CLDN18.2binds to the first extracellular loop of CLDN18.2. In one embodiment,the antibody having the ability of binding to CLDN18.2 mediates cellkilling by one or more of complement dependent cytotoxicity (CDC)mediated lysis, antibody dependent cellular cytotoxicity (ADCC) mediatedlysis, induction of apoptosis and inhibition of proliferation. In oneembodiment, the antibody having the ability of binding to CLDN18.2 is amonoclonal, chimeric or humanized antibody, or a fragment of anantibody. In one embodiment, the antibody having the ability of bindingto CLDN18.2 is an antibody selected from the group consisting of (i) anantibody produced by and/or obtainable from a clone deposited under theaccession no. DSM ACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSMACC2741, DSM ACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSMACC2747, DSM ACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) anantibody which is a chimerized or humanized form of the antibody under(i), (iii) an antibody having the specificity of the antibody under (i)and (iv) an antibody comprising the antigen binding portion or antigenbinding site, in particular the variable region, of the antibody under(i) and preferably having the specificity of the antibody under (i). Inone embodiment, the antibody is coupled to a therapeutic agent such as atoxin, a radioisotope, a drug or a cytotoxic agent.

In one embodiment, the method of the invention comprises administeringthe antibody having the ability of binding to CLDN18.2 at a dose of upto 1000 mg/m². In one embodiment, the method of the invention comprisesadministering the antibody having the ability of binding to CLDN18.2repeatedly at a dose of 300 to 600 mg/m².

In one embodiment, the cancer is CLDN18.2 positive. In one embodiment,the cancer disease is selected from the group consisting of gastriccancer, esophageal cancer, pancreatic cancer, lung cancer, ovariancancer, colon cancer, hepatic cancer, head-neck cancer, cancer of thegallbladder and the metastasis thereof. The cancer disease may be aKrukenberg tumor, peritoneal metastasis and/or lymph node metastasis. Inone embodiment, the cancer is an adenocarcinoma, in particular anadvanced adenocarcinoma. In one embodiment, the cancer is selected fromthe group consisting of cancer of the stomach, cancer of the esophagus,in particular the lower esophagus, cancer of the eso-gastric junctionand gastroesophageal cancer. The patient may be a HER2/neu negativepatient or a patient with HER2/neu positive status but not eligible totrastuzumab therapy.

According to the invention, CLDN18.2 preferably has the amino acidsequence according to SEQ ID NO: 1.

In a further aspect, the present invention provides a medicalpreparation comprising an antibody having the ability of binding toCLDN18.2 and an agent stimulating γδ T cells. The medical preparation ofthe present invention may further comprise an agent stabilizing orincreasing expression of CLDN18.2. The antibody having the ability ofbinding to CLDN18.2 and the agent stimulating γδ T cells, and optionallythe agent stabilizing or increasing expression of CLDN18.2, may bepresent in the medical preparation in a mixture or separate from eachother. The medical preparation may be a kit comprising a first containerincluding the antibody having the ability of binding to CLDN18.2 and acontainer including the agent stimulating γδ T cells, and optionally acontainer including the agent stabilizing or increasing expression ofCLDN18.2. The medical preparation may further include printedinstructions for use of the preparation for treatment of cancer, inparticular for use of the preparation in a method of the invention.Different embodiments of the medical preparation, and, in particular, ofthe agent stimulating γδ T cells and the agent stabilizing or increasingexpression of CLDN18.2 are as described above for the method of theinvention.

The present invention also provides the agents described herein such asthe antibody having the ability of binding to CLDN18.2 for use in themethods described herein, e.g. for administration in combination with anagent stimulating γδ T cells, and optionally an agent stabilizing orincreasing expression of CLDN18.2.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the effect of chemotherapyon gastric cancer cells. Cultivation of KatoIII cells for 96 hours leadsto a cell cycle arrest in the G0/G1-Phase and downregulation ofCLDN18.2. Cytostatic compounds resulting in a cell cycle arrest indifferent phases of the cell cycle (S-phase (5-FU) or G2-phase(epirubicin)) stabilize CLDN18.2-expression.

FIG. 2A, FIG. 2B, and FIG. 2C show the effect of chemotherapy on gastriccancer cells. FIG. 2A and FIG. 2B: Effect of chemotherapy on transcriptand protein levels of CLDN18.2 in gastric cancer cells. FIG. 2C: Flowcytometry of extracellular IMAB362 binding on gastric cancer cellstreated with chemotherapeutic agents

FIG. 3 shows the effect of chemotherapy on gastric cancer cells.Cytostatic compounds resulting in a cell cycle arrest in differentphases of the cell cycle (S/G2-phase (Irinotecan) or G2-phase(Docetaxel)).

FIG. 4A and FIG. 4B show IMAB362-induced ADCC mediated killing ofgastric cancer cells after pretreatment with chemotherapeutic agents

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D show the effect of chemotherapyon gastric cancer cells. FIG. 5A: Cells treated with Irinotecan,Docetaxel or Cisplatin exhibit a lower level of viable cells compared tomedium cultivated target cells. FIG. 5B: CLDN18.2 expression in cellstreated with Irinotecan, Docetaxel or Cisplatin is increased compared tomedium cultivated cells. FIG. 5C and FIG. 5D: Treatment of cells withIrinotecan, Docetaxel or Cisplatin augments the potency of IMAB362 toinduce ADCC.

FIG. 6 shows the effects of chemotherapy on IMAB362-induced CDC

FIG. 7A and FIG. 7B show the effects of chemotherapy on effector cells

FIG. 8A and FIG. 8B show the expansion of PBMCs in ZA/IL-2 supplementedcultures

FIG. 9A, FIG. 9B, and FIG. 9C show the enrichment of Vγ9Vδ2 T cells inZA/IL-2 supplemented PBMC cultures

FIG. 10 shows the enrichment of Vγ9Vδ2 T cells in medium supplementedwith ZA and an increasing IL-2 dose

FIG. 11A, FIG. 11B, and FIG. 11C show the expansion and cytotoxicactivity of Vγ9Vδ2 T cells upon co-incubation with ZA-pulsed monocytesand human cancer cells

FIG. 12 shows ZA-dependent development of different cell types in PBMCcultures

FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D show display of surfacemarkers on Vγ9Vδ2 T-cells after ZA/IL-2 treatment

FIG. 14A, FIG. 14B, and FIG. 14C show ADCC activity of Vγ9Vδ2 T cellswith IMAB362 on CLDN18.2-positive NUGC-4 gastric cancer cells

FIG. 15A, FIG. 15B, and FIG. 15C show ADCC of IMAB362 using Vγ9Vδ2 Tcells as effector cells

FIG. 16 shows the effects of ZA on surface localization of CLDN18.2 ontarget cells

FIG. 17 shows the effects of chemotherapy and ZA/IL-2 treatment oneffector cells

FIG. 18 shows biodistribution studies with conjugated antibodies in mice

FIG. 19 shows early treatment of HEK293˜CLDN18.2 tumor xenografts

FIG. 20 shows treatment of advanced HEK293˜CLDN18.2 tumor xenografts

FIG. 21A and FIG. 21B show the effect of IMAB362 on subcutaneous tumorgrowth of gastric cancer xenografts

FIG. 22A and FIG. 22B show the effects of immunotherapy with IMAB362 onNCI-N87˜CLDN18.2 gastric carcinoma xenografts

FIG. 23A and FIG. 23B show the effects of combination therapy withIMAB362 and EOF regimen on NCI-N87˜CLDN18.2 xenografts

FIG. 24A and FIG. 24B show the effects of combination therapy withIMAB362 and EOF regimen on NUGC-4˜CLDN18.2 xenografts

FIG. 25 shows the effect of ZA/IL-2 induced Vγ9Vδ2 T cells on control ofmacroscopic tumors by IMAB362 in NSG mice

FIG. 26 shows the effects of combination therapy with IMAB362 and EOFregimen on CLS-103˜cldn18.2 allograft tumors

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The term “CLDN18” relates to claudin 18 and includes any variants,including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) andclaudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

The term “CLDN18.2” preferably relates to human CLDN18.2, and, inparticular, to a protein comprising, preferably consisting of the aminoacid sequence according to SEQ ID NO: 1 of the sequence listing or avariant of said amino acid sequence.

The term “CLDN18.1” preferably relates to human CLDN18.1, and, inparticular, to a protein comprising, preferably consisting of the aminoacid sequence according to SEQ ID NO: 2 of the sequence listing or avariant of said amino acid sequence.

The term “variant” according to the invention refers, in particular, tomutants, splice variants, conformations, isoforms, allelic variants,species variants and species homologs, in particular those which arenaturally present. An allelic variant relates to an alteration in thenormal sequence of a gene, the significance of which is often unclear.Complete gene sequencing often identifies numerous allelic variants fora given gene. A species homolog is a nucleic acid or amino acid sequencewith a different species of origin from that of a given nucleic acid oramino acid sequence. The term “variant” shall encompass anyposttranslationally modified variants and conformation variants.

According to the invention, the term “CLDN18.2 positive cancer” means acancer involving cancer cells expressing CLDN18.2, preferably on thesurface of said cancer cells.

“Cell surface” is used in accordance with its normal meaning in the art,and thus includes the outside of the cell which is accessible to bindingby proteins and other molecules.

CLDN18.2 is expressed on the surface of cells if it is located at thesurface of said cells and is accessible to binding by CLDN18.2-specificantibodies added to the cells.

According to the invention, CLDN18.2 is not substantially expressed in acell if the level of expression is lower compared to expression instomach cells or stomach tissue. Preferably, the level of expression isless than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%of the expression in stomach cells or stomach tissue or even lower.Preferably, CLDN18.2 is not substantially expressed in a cell if thelevel of expression exceeds the level of expression in non-canceroustissue other than stomach by no more than 2-fold, preferably 1.5-fold,and preferably does not exceed the level of expression in saidnon-cancerous tissue. Preferably, CLDN18.2 is not substantiallyexpressed in a cell if the level of expression is below the detectionlimit and/or if the level of expression is too low to allow binding byCLDN18.2-specific antibodies added to the cells.

According to the invention, CLDN18.2 is expressed in a cell if the levelof expression exceeds the level of expression in non-cancerous tissueother than stomach preferably by more than 2-fold, preferably 10-fold,100-fold, 1000-fold, or 10000-fold. Preferably, CLDN18.2 is expressed ina cell if the level of expression is above the detection limit and/or ifthe level of expression is high enough to allow binding byCLDN18.2-specific antibodies added to the cells. Preferably, CLDN18.2expressed in a cell is expressed or exposed on the surface of said cell.

According to the invention, the term “disease” refers to anypathological state, including cancer, in particular those forms ofcancer described herein. Any reference herein to cancer or particularforms of cancer also includes cancer metastasis thereof. In a preferredembodiment, a disease to be treated according to the present applicationinvolves cells expressing CLDN18.2.

“Diseases associated with cells expressing CLDN18.2” or similarexpressions means according to the invention that CLDN18.2 is expressedin cells of a diseased tissue or organ. In one embodiment, expression ofCLDN18.2 in cells of a diseased tissue or organ is increased compared tothe state in a healthy tissue or organ. An increase refers to anincrease by at least 10%, in particular at least 20%, at least 50%, atleast 100%, at least 200%, at least 500%, at least 1000%, at least10000% or even more. In one embodiment, expression is only found in adiseased tissue, while expression in a healthy tissue is repressed.According to the invention, diseases associated with cells expressingCLDN18.2 include cancer diseases. Furthermore, according to theinvention, cancer diseases preferably are those wherein the cancer cellsexpress CLDN18.2.

As used herein, a “cancer disease” or “cancer” includes a diseasecharacterized by aberrantly regulated cellular growth, proliferation,differentiation, adhesion, and/or migration. By “cancer cell” is meantan abnormal cell that grows by a rapid, uncontrolled cellularproliferation and continues to grow after the stimuli that initiated thenew growth cease. Preferably, a “cancer disease” is characterized bycells expressing CLDN18.2 and a cancer cell expresses CLDN18.2. A cellexpressing CLDN18.2 preferably is a cancer cell, preferably of thecancers described herein.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so. Ovarianadenocarcinoma is the most common type of ovarian carcinoma. It includesthe serous and mucinous adenocarcinomas, the clear cell adenocarcinomaand the endometrioid adenocarcinoma.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system. In one embodiment, the term “metastasis” according tothe invention relates to lymph node metastasis. One particular form ofmetastasis which is treatable using the therapy of the invention ismetastasis originating from gastric cancer as primary site. In preferredembodiments such gastric cancer metastasis is Krukenberg tumors,peritoneal metastasis and/or lymph node metastasis.

Krukenberg tumor is an uncommon metastatic tumor of the ovary accountingfor 1% to 2% of all ovarian tumors. Prognosis of Krukenberg tumor isstill very poor and there is no established treatment for Krukenbergtumors. Krukenberg tumor is a metastatic signet ring cell adenocarcinomaof the ovary. Stomach is the primary site in most Krukenberg tumor cases(70%). Carcinomas of colon, appendix, and breast (mainly invasivelobular carcinoma) are the next most common primary sites. Rare cases ofKrukenberg tumor originating from carcinomas of the gallbladder, biliarytract, pancreas, small intestine, ampulla of Vater, cervix, and urinarybladder/urachus have been reported. The interval between the diagnosisof a primary carcinoma and the subsequent discovery of ovarianinvolvement is usually 6 months or less, but longer periods have beenreported. In many cases, the primary tumor is very small and can escapedetection. A history of a prior carcinoma of the stomach or anotherorgan can be obtained in only 20% to 30% of the cases.

Krukenberg tumor is an example of the selective spread of cancers, mostcommonly in the stomach-ovarian axis. This axis of tumor spread hashistorically drawn the attention of many pathologists, especially whenit was found that gastric neoplasms selectively metastasize to theovaries without involvement of other tissues. The route of metastasis ofgastric carcinoma to the ovaries has been a mystery for a long time, butit is now evident that retrograde lymphatic spread is the most likelyroute of metastasis.

Women with Krukenberg tumors tend to be unusually young for patientswith metastatic carcinoma as they are typically in the fifth decade oftheir lives, with an average age of 45 years. This young age ofdistribution can be related in part to the increased frequency ofgastric signet ring cell carcinomas in young women. Common presentingsymptoms are usually related to ovarian involvement, the most common ofwhich are abdominal pain and distension (mainly because of the usuallybilateral and often large ovarian masses). The remaining patients havenonspecific gastrointestinal symptoms or are asymptomatic. In addition,Krukenberg tumor is reportedly associated with virilization resultingfrom hormone production by ovarian stroma. Ascites is present in 50% ofthe cases and usually reveals malignant cells.

Krukenberg tumors are bilateral in more than 80% of the reported cases.The ovaries are usually asymmetrically enlarged, with a bosselatedcontour. The sectioned surfaces are yellow or white; they are usuallysolid, although they are occasionally cystic. Importantly, the capsularsurface of the ovaries with Krukenberg tumors is typically smooth andfree of adhesions or peritoneal deposits. Of note, other metastatictumors to the ovary tend to be associated with surface implants. Thismay explain why the gross morphology of Krukenberg tumor can deceptivelyappear as a primary ovarian tumor. However, bilateralism in Krukenbergtumor is consistent with its metastatic nature.

Patients with Krukenberg tumors have an overall mortality rate that issignificantly high. Most patients die within 2 years (median survival,14 months). Several studies show that the prognosis is poor when theprimary tumor is identified after the metastasis to the ovary isdiscovered, and the prognosis becomes worse if the primary tumor remainscovert.

No optimal treatment strategy for Krukenberg tumors has been clearlyestablished in the literature. Whether a surgical resection should beperformed has not been adequately addressed. Chemotherapy orradiotherapy has no significant effect on prognosis of patients withKrukenberg tumors.

By “treat” is meant to administer a compound or composition or acombination of compounds or compositions to a subject in order toprevent or eliminate a disease, including reducing the size of a tumoror the number of tumors in a subject; arrest or slow a disease in asubject; inhibit or slow the development of a new disease in a subject;decrease the frequency or severity of symptoms and/or recurrences in asubject who currently has or who previously has had a disease; and/orprolong, i.e. increase the lifespan of the subject.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, preventing, slowing down orinhibiting progression or worsening, or preventing or delaying the onsetof a disease or the symptoms thereof.

The term “patient” means according to the invention a subject fortreatment, in particular a diseased subject, including human beings,nonhuman primates or another animals, in particular mammals such ascows, horses, pigs, sheeps, goats, dogs, cats or rodents such as miceand rats. In a particularly preferred embodiment, a patient is a humanbeing. γδ T cells (gamma delta T cells) represent a small subset of Tcells that possess a distinct T cell receptor (TCR) on their surface. Amajority of T cells have a TCR composed of two glycoprotein chainscalled α- and β-TCR chains. In contrast, in γδ T cells, the TCR is madeup of one γ-chain and one δ-chain. This group of T cells is usually muchless common than αβT cells. Human γδ T cells play an important role instress-surveillance responses like infectious diseases and autoimmunity.Transformation-induced changes in tumors are also suggested to causestress-surveillance responses mediated by γδ T cells and enhanceantitumor immunity. Importantly, after antigen engagement, activated γδT cells at lesional sites provide cytokines (e.g. INFγ, TNFα) and/orchemokines mediating recruitment of other effector cells and showimmediate effector functions such as cytotoxicity (via death receptorand cytolytic granules pathways) and ADCC.

The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cellreceptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates andare assumed to play an early and essential role in sensing “danger” byinvading pathogens as they expand dramatically in many acute infectionsand may exceed all other lymphocytes within a few days, e.g. intuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia,listeriosis, toxoplasmosis, and malaria.

γδ T cells respond to small non-peptidic phosphorylated antigens(phosphoantigens) such as pyrophosphates synthesized in bacteria andisopentenyl pyrophosphate (IPP) produced in mammalian cells through themevalonate pathway. Whereas IPP production in normal cells is notsufficient for activation of γδ T cells, dysregulation of the mevalonatepathway in tumor cells leads to accumulation of IPP and γδ T cellactivation. IPPs can also be therapeutically increased byaminobisphosphonates, which inhibit the mevalonate pathway enzymefarnesyl pyrophosphate synthase (FPPS). Among others, zoledronic acid(ZA, zoledronate, Zometa™, Novartis) represents such anaminobiphosphonate, which is already clinically administered to patientsfor the treatment of osteoporosis and metastasic bone disease. Upontreatment of PBMCs in vitro, ZA is taken up especially by monocytes. IPPaccumulates in the monocytes and they differentiate toantigen-presenting cells stimulating development of γδ T cells. In thissetting, the addition of interleukin-2 (IL-2) is preferred as growth andsurvival factor for activated γδ T cells. Finally, certain alkylatedamines have been described to activate Vγ9Vδ2 T cells in vitro, howeveronly at millimolar concentrations.

According to the invention, the term “agent stimulating γδ T cells”relates to compounds stimulating development of γδ T cells, inparticular Vγ9Vδ2 T cells, in vitro and/or in vivo, in particular byinducing activation and expansion of γδ T cells. Preferably, the termrelates to compounds which in vitro and/or in vivo increase isopentenylpyrophosphate (IPP) produced in mammalian cells, preferably byinhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase(FPPS).

One particular group of compounds stimulating γδ T cells arebisphosphonates, in particular nitrogen-containing bisphosphonates(N-bisphosphonates; aminobisphosphonates).

For example, suitable bisphosphonates for use in the invention mayinclude one or more of the following compounds including analogs andderivatives, pharmaceutical salts, hydrates, esters, conjugates andprodrugs thereof:

-   [1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid),    zoledronic acid, e.g. zoledronate;-   (dichloro-phosphono-methyl)phosphonic acid, e.g. clodronate-   {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic    acid), ibandronic acid, e.g. ibandronate-   (3-amino-1-hydroxypropane-1,1-diyl)bis (phosphonic acid), pamidronic    acid, e.g. pamidronate;-   (1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid,    risedronic acid, e.g. risedronate;-   (1-Hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic    acid, minodronic acid;-   [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid),    olpadronic acid.-   [4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic    acid, alendronic acid, e.g. alendronate;-   [(Cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid;-   (1-hydroxyethan-1,1-diyl)bis(phosphonic acid), etidronic acid, e.g.    etidronate; and-   {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic    acid.

According to the invention, zoledronic acid (INN) or zoledronate(marketed by Novartis under the trade names Zometa, Zomera, Aclasta andReclast) is a particularly preferred bisphosphonate. Zometa is used toprevent skeletal fractures in patients with cancers such as multiplemyeloma and prostate cancer, as well as for treating osteoporosis. Itcan also be used to treat hypercalcemia of malignancy and can be helpfulfor treating pain from bone metastases.

In one particularly preferred embodiment, an agent stimulating γδ Tcells according to the invention is administered in combination withIL-2. Such combination has been shown to be particularly effective inmediating expansion and activation of γ9δ2 T cells.

Interleukin-2 (IL-2) is an interleukin, a type of cytokine signalingmolecule in the immune system. It is a protein that attracts lymphocytesand is part of the body's natural response to microbial infection, andin discriminating between foreign (non-self) and self. IL-2 mediates itseffects by binding to IL-2 receptors, which are expressed bylymphocytes.

The IL-2 used according to the invention may be any IL-2 supporting orenabling the stimulation of γδ T cells and may be derived from anyspecies, preferably human. Il-2 may be isolated, recombinantly producedor synthetic IL-2 and may be naturally occurring or modified IL-2.

The term “agent stabilizing or increasing expression of CLDN18.2” refersto an agent or a combination of agents the provision of which to cellsresults in increased RNA and/or protein levels of CLDN18.2, preferablyin increased levels of CLDN18.2 protein on the cell surface, compared tothe situation where the cells are not provided with the agent or thecombination of agents. Preferably, the cell is a cancer cell, inparticular a cancer cell expressing CLDN18.2, such as a cell of thecancer types described herein. The term “agent stabilizing or increasingexpression of CLDN18.2” refers, in particular, to an agent or acombination of agents the provision of which to cells results in ahigher density of CLDN18.2 on the surface of said cells compared to thesituation where the cells are not provided with the agent or thecombination of agents. “Stabilizing expression of CLDN18.2” includes, inparticular, the situation where the agent or the combination of agentsprevents a decrease or reduces a decrease in expression of CLDN18.2,e.g. expression of CLDN18.2 would decrease without provision of theagent or the combination of agents and provision of the agent or thecombination of agents prevents said decrease or reduces said decrease ofCLDN18.2 expression. “Increasing expression of CLDN18.2” includes, inparticular, the situation where the agent or the combination of agentsincreases expression of CLDN18.2, e.g. expression of CLDN18.2 woulddecrease, remain essentially constant or increase without provision ofthe agent or the combination of agents and provision of the agent or thecombination of agents increases CLDN18.2 expression compared to thesituation without provision of the agent or the combination of agents sothat the resulting expression is higher compared to the situation whereexpression of CLDN18.2 would decrease, remain essentially constant orincrease without provision of the agent or the combination of agents.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” includes chemotherapeutic agents or combinationsof chemotherapeutic agents such as cytostatic agents. Chemotherapeuticagents may affect cells in one of the following ways: (1) Damage the DNAof the cells so they can no longer reproduce, (2) Inhibit the synthesisof new DNA strands so that no cell replication is possible, (3) Stop themitotic processes of the cells so that the cells cannot divide into twocells.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” preferably relates to an agent or a combinationof agents such a cytostatic compound or a combination of cytostaticcompounds the provision of which to cells, in particular cancer cells,results in the cells being arrested in or accumulating in one or morephases of the cell cycle, preferably in one or more phases of the cellcycle other than the G1- and G0-phases, preferably other than theG1-phase, preferably in one or more of the G2- or S-phase of the cellcycle such as the G1/G2-, S/G2-, G2- or S-phase of the cell cycle. Theterm “cells being arrested in or accumulating in one or more phases ofthe cell cycle” means that the precentage of cells which are in said oneor more phases of the cell cycle increases. Each cell goes through acycle comprising four phases in order to replicate itself. The firstphase called G1 is when the cell prepares to replicate its chromosomes.The second stage is called S, and in this phase DNA synthesis occurs andthe DNA is duplicated. The next phase is the G2 phase, when the RNA andprotein duplicate. The final stage is the M stage, which is the stage ofactual cell division. In this final stage, the duplicated DNA and RNAsplit and move to separate ends of the cell, and the cell actuallydivides into two identical, functional cells. Chemotherapeutic agentswhich are DNA damaging agents usually result in an accumulation of cellsin the G1 and/or G2 phase. Chemotherapeutic agents which block cellgrowth by interfering with DNA synthesis such as antimetabolites usuallyresult in an accumulation of cells in the S-phase. Examples of thesedrugs are 6-mercaptopurine and 5-fluorouracil.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” includes anthracyclines such as epirubicin,platinum compounds such as oxaliplatin and cisplatin, nucleoside analogssuch as 5-fluorouracil or prodrugs thereof, taxanes such as docetaxel,and camptothecin analogs such as irinotecan and topotecan, andcombinations of drugs such as combinations of drugs comprising one ormore of anthracyclines such as epirubicin, oxaliplatin and5-fluorouracil such as a combination of drugs comprising oxaliplatin and5-fluorouracil or other drug combinations described herein.

In one preferred embodiment, an “agent stabilizing or increasingexpression of CLDN18.2” is an “agent inducing immunogenic cell death”.

In specific circumstances, cancer cells can enter a lethal stresspathway linked to the emission of a spatiotemporally defined combinationof signals that is decoded by the immune system to activatetumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:798-804). In such scenario cancer cells are triggered to emit signalsthat are sensed by innate immune effectors such as dendritic cells totrigger a cognate immune response that involves CD8+ T cells and IFN-γsignalling so that tumor cell death may elicit a productive anticancerimmune response. These signals include the pre-apoptotic exposure of theendoplasmic reticulum (ER) chaperon calreticulin (CRT) at the cellsurface, the pre-apoptotic secretion of ATP, and the post-apoptoticrelease of the nuclear protein HMGB1. Together, these processesconstitute the molecular determinants of immunogenic cell death (ICD).Anthracyclines, oxaliplatin, and γ irradiation are able to induce allsignals that define ICD, while cisplatin, for example, which isdeficient in inducing CRT translocation from the ER to the surface ofdying cells—a process requiring ER stress—requires complementation bythapsigargin, an ER stress inducer.

According to the invention, the term “agent inducing immunogenic celldeath” refers to an agent or a combination of agents which when providedto cells, in particular cancer cells, is capable of inducing the cellsto enter a lethal stress pathway which finally results in tumor-specificimmune responses. In particular, an agent inducing immunogenic celldeath when provided to cells induces the cells to emit aspatiotemporally defined combination of signals, including, inparticular, the pre-apoptotic exposure of the endoplasmic reticulum (ER)chaperon calreticulin (CRT) at the cell surface, the pre-apoptoticsecretion of ATP, and the post-apoptotic release of the nuclear proteinHMGB1.

According to the invention, the term “agent inducing immunogenic celldeath” includes anthracyclines and oxaliplatin.

Anthracyclines are a class of drugs commonly used in cancer chemotherapythat are also antibiotics. Structurally, all anthracyclines share acommon four-ringed 7,8,9,10-tetrahydrotetracene-5,12-quinone structureand usually require glycosylation at specific sites.

Anthracyclines preferably bring about one or more of the followingmechanisms of action: 1. Inhibiting DNA and RNA synthesis byintercalating between base pairs of the DNA/RNA strand, thus preventingthe replication of rapidly-growing cancer cells. 2. Inhibitingtopoisomerase II enzyme, preventing the relaxing of supercoiled DNA andthus blocking DNA transcription and replication. 3. Creatingiron-mediated free oxygen radicals that damage the DNA and cellmembranes.

According to the invention, the term “anthracycline” preferably relatesto an agent, preferably an anticancer agent for inducing apoptosis,preferably by inhibiting the rebinding of DNA in topoisomerase II.

Preferably, according to the invention, the term “anthracycline”generally refers to a class of compounds having the following ringstructure

including analogs and derivatives, pharmaceutical salts, hydrates,esters, conjugates and prodrugs thereof.

Examples of anthracyclines and anthracycline analogs include, but arenot limited to, daunorubicin (daunomycin), doxorubicin (adriamycin),epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin,N-trifluoro-acetyl doxorubicin-14-valerate, aclacinomycin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX),5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclarubicin).Mitoxantrone is a member of the anthracendione class of compounds, whichare anthracycline analogs that lack the sugar moiety of theanthracyclines but retain the planar polycylic aromatic ring structurethat permits intercalation into DNA.

Particularly preferred as anthracyline according to the invention is acompound of the following formula:

whereinR₁ is selected from the group consisting of H and OH, R₂ is selectedfrom the group consisting of H and OMe, R₃ is selected from the groupconsisting of H and OH, and R₄ is selected from the group consisting ofH and OH.

In one embodiment, R₁ is H, R₂ is OMe, R₃ is H, and R₄ is OH. In anotherembodiment, R₁ is OH, R₂ is OMe, R₃ is H, and R₄ is OH. In anotherembodiment, R₁ is OH, R₂ is OMe, R₃ is OH, and R₄ is H. In anotherembodiment, R₁ is H, R₂ is H, R₃ is H, and R₄ is OH.

Specifically contemplated as anthracycline in the context of the presentinvention is epirubicin. Epirubicin is an anthracycline drug which hasthe following formula:

and is marketed under the trade name Ellence in the US and Pharmorubicinor Epirubicin Ebewe elsewhere. In particular, the term “epirubicin”refers to the compound(8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracen-5,12-dion.Epirubicin is favoured over doxorubicin, the most popular anthracycline,in some chemotherapy regimens as it appears to cause fewer side-effects.

According to the invention, the term “platinum compound” refers tocompounds containing platinum in their structure such as platinumcomplexes and includes compounds such as cisplatin, carboplatin andoxaliplatin.

The term “cisplatin” or “cisplatinum” refers to the compoundcis-diamminedichloroplatinum(II) (CDDP) of the following formula:

The term “carboplatin” refers to the compoundcis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) of the followingformula:

The term “oxaliplatin” refers to a compound which is a platinum compoundthat is complexed to a diaminocyclohexane carrier ligand of thefollowing formula:

In particular, the term “oxaliplatin” refers to the compound[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II).Oxaliplatin for injection is also marketed under the trade nameEloxatine.

The term “nucleoside analog” refers to a structural analog of anucleoside, a category that includes both purine analogs and pyrimidineanalogs. In particular, the term “nucleoside analog” refers tofluoropyrimidine derivatives which includes fluorouracil and prodrugsthereof.

The term “fluorouracil” or “5-fluorouracil” (5-FU or f5U) (sold underthe brand names Adrucil, Carac, Efudix, Efudex and Fluoroplex) is acompound which is a pyrimidine analog of the following formula:

In particular, the term refers to the compound5-fluoro-1H-pyrimidine-2,4-dione.

The term “capecitabine” (Xeloda, Roche) refers to a chemotherapeuticagent that is a prodrug that is converted into 5-FU in the tissues.Capecitabine which may be orally administered has the following formula:

In particular, the term refers to the compoundpentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

Taxanes are a class of diterpene compounds that were first derived fromnatural sources such as plants of the genus Taxus, but some have beensynthesized artificially. The principal mechanism of action of thetaxane class of drugs is the disruption of microtubule function, therebyinhibiting the process of cell division. Taxanes include docetaxel(Taxotere) and paclitaxel (Taxol).

According to the invention, the term “docetaxel” refers to a compoundhaving the following formula:

According to the invention, the term “paclitaxel” refers to a compoundhaving the following formula:

According to the invention, the term “camptothecin analog” refers toderivatives of the compound camptothecin (CPT;(S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′: 6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione). Preferably, the term “camptothecinanalog” refers to compounds comprising the following structure:

According to the invention, preferred camptothecin analogs areinhibitors of DNA enzyme topoisomerase I (topo I). Preferredcamptothecin analogs according to the invention are irinotecan andtopotecan.

Irinotecan is a drug preventing DNA from unwinding by inhibition oftopoisomerase I. In chemical terms, it is a semisynthetic analogue ofthe natural alkaloid camptothecin having the following formula:

In particular, the term “irinotecan” refers to the compound(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate.

Topotecan is a topoisomerase inhibitor of the formula:

In particular, the term “topotecan” refers to the compound(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionemonohydrochloride.

According to the invention, an agent stabilizing or increasingexpression of CLDN18.2 may be a chemotherapeutic agent, in particular achemotherapeutic agent established in cancer treatment and may be partof a combination of drugs such as a combination of drugs established foruse in cancer treatment. Such combination of drugs may be a drugcombination used in chemotherapy, and may be a drug combination as usedin a chemotherapeutic regimen selected from the group consisting of EOXchemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLOchemotherapy, FOLFOX chemotherapy, FOLFIRI chemotherapy, DCFchemotherapy and FLOT chemotherapy.

The drug combination used in EOX chemotherapy comprises of epirubicin,oxaliplatin and capecitabine. The drug combination used in ECFchemotherapy comprises of epirubicin, cisplatin and 5-fluorouracil. Thedrug combination used in ECX chemotherapy comprises of epirubicin,cisplatin and capecitabine. The drug combination used in EOFchemotherapy comprises of epirubicin, oxaliplatin and 5-fluorouracil.

Epirubicin is normally given at a dose of 50 mg/m2, cisplatin 60 mg/m2,oxaliplatin 130 mg/m2, protracted venous infusion of 5-fluorouracil at200 mg/m2/day and oral capecitabine 625 mg/m2 twice daily, for a totalof eight 3-week cycles.

The drug combination used in FLO chemotherapy comprises of5-fluorouracil, folinic acid and oxaliplatin (normally 5-fluorouracil2,600 mg/m2 24-h infusion, folinic acid 200 mg/m2 and oxaliplatin 85mg/m2, every 2 weeks).

FOLFOX is a chemotherapy regimen made up of folinic acid (leucovorin),5-fluorouracil and oxaliplatin. The recommended dose schedule givenevery two weeks is as follows: Day 1: Oxaliplatin 85 mg/m² IV infusionand leucovorin 200 mg/m² IV infusion, followed by 5-FU 400 mg/m² IVbolus, followed by 5-FU 600 mg/m² IV infusion as a 22-hour continuousinfusion; Day 2: Leucovorin 200 mg/m² IV infusion over 120 minutes,followed by 5-FU 400 mg/m² IV bolus given over 2-4 minutes, followed by5-FU 600 mg/m² IV infusion as a 22-hour continuous infusion.

The drug combination used in FOLFIRI chemotherapy comprises of5-fluorouracil, leucovorin, and irinotecan.

The drug combination used in DCF chemotherapy comprises of docetaxel,cisplatin and 5-fluorouracil.

The drug combination used in FLOT chemotherapy comprises of docetaxel,oxaliplatin, 5-fluorouracil and folinic acid.

The term “folinic acid” or “leucovorin” refers to a compound useful insynergistic combination with the chemotherapy agent 5-fluorouracil.Folinic acid has the following formula:

In particular, the term refers to the compound(2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}pentanedioicacid.

For administering an agent stabilizing or increasing expression ofCLDN18.2, in one embodiment, the standard chemotherapy according to theEOX regimen in combination with an antibody having the ability ofbinding to CLDN18.2, in particular IMAB362 is administered for max. 8cycles. The doses and schedules may be as follows:

-   -   50 mg/m2 Epirubicin will be administered i.v. as 15 minute        infusion on day 1 of each cycle during the EOX phase.    -   130 mg/m2 Oxaliplatin will be administered i.v. as 2 h infusion        on day 1 of each cycle during the EOX phase.    -   625 mg/m2 Capecitabine are taken p.o. twice daily for 21 days in        the morning and in the evening starting with the evening of day        1 of each cycle during the EOX phase.    -   1000 mg/m2 antibody will be administered i.v. as a 2 h infusion        on day 1 of cycle 1. Thereafter 600 mg/m2 antibody will be        administered i.v. as 2 h infusion on day 1 of each other cycle        after infusion of Oxaliplatin is completed.    -   After termination of chemotherapy, the patient will continue        with 600 mg/m2 antibody as 2 h infusion every 3 or 4 weeks.

In one embodiment of the present invention, the standard chemotherapyaccording to the EOX regimen in combination with ZA/IL-2 and an antibodyhaving the ability of binding to CLDN18.2, in particular IMAB362 isadministered for up to 8 cycles (24 weeks).

The term “antigen” relates to an agent such as a protein or peptidecomprising an epitope against which an immune response is directedand/or is to be directed. In a preferred embodiment, an antigen is atumor-associated antigen, such as CLDN18.2, i.e., a constituent ofcancer cells which may be derived from the cytoplasm, the cell surfaceand the cell nucleus, in particular those antigens which are produced,preferably in large quantity, intracellular or as surface antigens oncancer cells.

In the context of the present invention, the term “tumor-associatedantigen” preferably relates to proteins that are under normal conditionsspecifically expressed in a limited number of tissues and/or organs orin specific developmental stages and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In the context of thepresent invention, the tumor-associated antigen is preferably associatedwith the cell surface of a cancer cell and is preferably not or onlyrarely expressed in normal tissues.

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. An epitope of a protein such as CLDN18.2 preferablycomprises a continuous or discontinuous portion of said protein and ispreferably between 5 and 100, preferably between 5 and 50, morepreferably between 8 and 30, most preferably between 10 and 25 aminoacids in length, for example, the epitope may be preferably 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids in length.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, and includes any molecule comprising an antigen binding portionthereof. The term “antibody” includes monoclonal antibodies andfragments or derivatives of antibodies, including, without limitation,human antibodies, humanized antibodies, chimeric antibodies, singlechain antibodies, e.g., scFv's and antigen-binding antibody fragmentssuch as Fab and Fab′ fragments and also includes all recombinant formsof antibodies, e.g., antibodies expressed in prokaryotes, unglycosylatedantibodies, and any antigen-binding antibody fragments and derivativesas described herein. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies described herein may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals, while the constant portions are homologous to sequences ofantibodies derived from another. One clear advantage to such chimericforms is that the variable region can conveniently be derived frompresently known sources using readily available B-cells or hybridomasfrom non-human host organisms in combination with constant regionsderived from, for example, human cell preparations. While the variableregion has the advantage of ease of preparation and the specificity isnot affected by the source, the constant region being human, is lesslikely to elicit an immune response from a human subject when theantibodies are injected than would the constant region from a non humansource. However the definition is not limited to this particularexample.

The terms “antigen-binding portion” of an antibody (or simply “bindingportion”) or “antigen-binding fragment” of an antibody (or simply“binding fragment”) or similar terms refer to one or more fragments ofan antibody that retain the ability to specifically bind to an antigen.It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. A further example is binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region. The binding-domainimmunoglobulin fusion proteins are further disclosed in US 2003/0118592and US 2003/0133939. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with (a) a cell surface antigen, and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to CLDN18.2, and to other targets, such as Fc receptors oneffector cells. The term “bispecific antibodies” also includesdiabodies. Diabodies are bivalent, bispecific antibodies in which the VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

An antibody may be conjugated to a therapeutic moiety or agent, such asa cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Acytotoxin or cytotoxic agent includes any agent that is detrimental toand, in particular, kills cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Suitable therapeutic agents for forming antibodyconjugates include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents(e.g., vincristine and vinblastine). In a preferred embodiment, thetherapeutic agent is a cytotoxic agent or a radiotoxic agent. In anotherembodiment, the therapeutic agent is an immunosuppressant. In yetanother embodiment, the therapeutic agent is GM-CSF. In a preferredembodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin,sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies also can be conjugated to a radioisotope, e.g., iodine-131,yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62: 119-58 (1982).

As used herein, an antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system by immunizing ananimal or by screening an immunoglobulin gene library, and wherein theselected antibody is at least 90%, more preferably at least 95%, evenmore preferably at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, an antibody derived from a particulargermline sequence will display no more than 10 amino acid differences,more preferably, no more than 5, or even more preferably, no more than4, 3, 2, or 1 amino acid difference from the amino acid sequence encodedby the germline immunoglobulin gene.

As used herein, the term “heteroantibodies” refers to two or moreantibodies, derivatives thereof, or antigen binding regions linkedtogether, at least two of which have different specificities. Thesedifferent specificities include a binding specificity for an Fc receptoron an effector cell, and a binding specificity for an antigen or epitopeon a target cell, e.g., a tumor cell.

The antibodies described herein may be monoclonal antibodies. The term“monoclonal antibody” as used herein refers to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodydisplays a single binding specificity and affinity. In one embodiment,the monoclonal antibodies are produced by a hybridoma which includes a Bcell obtained from a non-human animal, e.g., mouse, fused to animmortalized cell.

The antibodies described herein may be recombinant antibodies. The term“recombinant antibody”, as used herein, includes all antibodies that areprepared, expressed, created or isolated by recombinant means, such as(a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

Antibodies described herein may be derived from different species,including but not limited to mouse, rat, rabbit, guinea pig and human.

Antibodies described herein include polyclonal and monoclonal antibodiesand include IgA such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM,and IgD antibodies. In various embodiments, the antibody is an IgG1antibody, more particularly an IgG1, kappa or IgG1, lambda isotype (i.e.IgG1, κ, λ), an IgG2a antibody (e.g. IgG2a, κ, λ), an IgG2b antibody(e.g. IgG2b, κ, λ), an IgG3 antibody (e.g. IgG3, κ, λ) or an IgG4antibody (e.g. IgG4, κ, λ).

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The invention includes all antibodies and derivatives of antibodies asdescribed herein which for the purposes of the invention are encompassedby the term “antibody”. The term “antibody derivatives” refers to anymodified form of an antibody, e.g., a conjugate of the antibody andanother agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLDN18.2 is substantially free of antibodies that specifically bindantigens other than CLDN18.2). An isolated antibody that specificallybinds to an epitope, isoform or variant of human CLDN18.2 may, however,have cross-reactivity to other related antigens, e.g., from otherspecies (e.g., CLDN18.2 species homologs). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals. In one embodiment of the invention, a combination of“isolated” monoclonal antibodies relates to antibodies having differentspecificities and being combined in a well defined composition ormixture.

The term “binding” according to the invention preferably relates to aspecific binding.

According to the present invention, an antibody is capable of binding toa predetermined target if it has a significant affinity for saidpredetermined target and binds to said predetermined target in standardassays. “Affinity” or “binding affinity” is often measured byequilibrium dissociation constant (K_(D)). Preferably, the term“significant affinity” refers to the binding to a predetermined targetwith a dissociation constant (K_(D)) of 10⁻⁵M or lower, 10⁻⁶ M or lower,10⁻⁷ M or lower, 10⁻⁸ M or lower, 10⁻⁹ M or lower, 10⁻¹⁰ M or lower,10⁻¹¹ M or lower, or 10⁻¹²M or lower.

An antibody is not (substantially) capable of binding to a target if ithas no significant affinity for said target and does not bindsignificantly, in particular does not bind detectably, to said target instandard assays. Preferably, the antibody does not detectably bind tosaid target if present in a concentration of up to 2, preferably 10,more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably,an antibody has no significant affinity for a target if it binds to saidtarget with a K_(D) that is at least 10-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold, or 10⁶-fold higher than the K_(D) for binding to thepredetermined target to which the antibody is capable of binding. Forexample, if the K_(D) for binding of an antibody to the target to whichthe antibody is capable of binding is 10⁻⁷ M, the K_(D) for binding to atarget for which the antibody has no significant affinity would be is atleast 10⁻⁶ M, 10⁻⁵ M, 10⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

An antibody is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not capable of bindingto other targets, i.e. has no significant affinity for other targets anddoes not significantly bind to other targets in standard assays.According to the invention, an antibody is specific for CLDN18.2 if itis capable of binding to CLDN18.2 but is not (substantially) capable ofbinding to other targets. Preferably, an antibody is specific forCLDN18.2 if the affinity for and the binding to such other targets doesnot significantly exceed the affinity for or binding toCLDN18.2-unrelated proteins such as bovine serum albumin (BSA), casein,human serum albumin (HSA) or non-claudin transmembrane proteins such asMHC molecules or transferrin receptor or any other specifiedpolypeptide. Preferably, an antibody is specific for a predeterminedtarget if it binds to said target with a K_(D) that is at least 10-fold,100-fold, 10³-fold, 10⁴-fold, 10⁵-fold, or 10⁶-fold lower than the K_(D)for binding to a target for which it is not specific. For example, ifthe K_(D) for binding of an antibody to the target for which it isspecific is 10⁻⁷M, the K_(D) for binding to a target for which it is notspecific would be at least 10⁻⁶ M, 10⁻⁵ M, 10⁴ M, 10⁻³ M, 10⁻² M, or10⁻¹ M.

Binding of an antibody to a target can be determined experimentallyusing any suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N Y (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

According to the invention an antibody having the ability of binding toCLDN18.2 is an antibody capable of binding to an epitope present inCLDN18.2, preferably an epitope located within the extracellular domainsof CLDN18.2, in particular the first extracellular domain, preferablyamino acid positions 29 to 78 of CLDN18.2. In particular embodiments, anantibody having the ability of binding to CLDN18.2 is an antibodycapable of binding to (i) an epitope on CLDN18.2 which is not present onCLDN18.1, preferably SEQ ID NO: 3, 4, and 5, (ii) an epitope localizedon the CLDN18.2-loop1, preferably SEQ ID NO: 8, (iii) an epitopelocalized on the CLDN18.2-loop2, preferably SEQ ID NO: 10, (iv) anepitope localized on the CLDN18.2-loopD3, preferably SEQ ID NO: 11, (v)an epitope, which encompass CLDN18.2-loop1 and CLDN18.2-loopD3, or (vi)a non-glycosylated epitope localized on the CLDN18.2-loopD3, preferablySEQ ID NO: 9.

According to the invention an antibody having the ability of binding toCLDN18.2 preferably is an antibody having the ability of binding toCLDN18.2 but not to CLDN18.1. Preferably, an antibody having the abilityof binding to CLDN18.2 is specific for CLDN18.2. Preferably, an antibodyhaving the ability of binding to CLDN18.2 preferably is an antibodyhaving the ability of binding to CLDN18.2 expressed on the cell surface.In particular preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 binds to native epitopes of CLDN18.2 present on thesurface of living cells. Preferably, an antibody having the ability ofbinding to CLDN18.2 binds to one or more peptides selected from thegroup consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably,an antibody having the ability of binding to CLDN18.2 is specific forthe afore mentioned proteins, peptides or immunogenic fragments orderivatives thereof. An antibody having the ability of binding toCLDN18.2 may be obtained by a method comprising the step of immunizingan animal with a protein or peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and48-50, or a nucleic acid or host cell expressing said protein orpeptide. Preferably, the antibody binds to cancer cells, in particularcells of the cancer types mentioned above and, preferably, does not bindsubstantially to non-cancerous cells.

Preferably, binding of an antibody having the ability of binding toCLDN18.2 to cells expressing CLDN18.2 induces or mediates killing ofcells expressing CLDN18.2. The cells expressing CLDN18.2 are preferablycancer cells and are, in particular, selected from the group consistingof tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon,hepatic, head-neck, and gallbladder cancer cells. Preferably, theantibody induces or mediates killing of cells by inducing one or more ofcomplement dependent cytotoxicity (CDC) mediated lysis, antibodydependent cellular cytotoxicity (ADCC) mediated lysis, apoptosis, andinhibition of proliferation of cells expressing CLDN18.2. Preferably,ADCC mediated lysis of cells takes place in the presence of effectorcells, which in particular embodiments are selected from the groupconsisting of monocytes, mononuclear cells, NK cells and PMNs.Inhibiting proliferation of cells can be measured in vitro bydetermining proliferation of cells in an assay using bromodeoxyuridine(5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside which isan analogue of thymidine and can be incorporated into the newlysynthesized DNA of replicating cells (during the S phase of the cellcycle), substituting for thymidine during DNA replication. Detecting theincorporated chemical using, for example, antibodies specific for BrdUindicates cells that were actively replicating their DNA.

In preferred embodiments, antibodies described herein can becharacterized by one or more of the following properties:

-   a) specificity for CLDN18.2;-   b) a binding affinity to CLDN18.2 of about 100 nM or less,    preferably, about 5-10 nM or less and, more preferably, about 1-3 nM    or less,-   c) the ability to induce or mediate CDC on CLDN18.2 positive cells;-   d) the ability to induce or mediate ADCC on CLDN18.2 positive cells;-   e) the ability to inhibit the growth of CLDN18.2 positive cells;-   f) the ability to induce apoptosis of CLDN18.2 positive cells.

In a particularly preferred embodiment, an antibody having the abilityof binding to CLDN18.2 is produced by a hybridoma deposited at the DSMZ(Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address:Inhoffenstr. 7B, 31824 Braunschweig, Germany) and having the followingdesignation and accession number:

-   a. 182-D1106-055, accession no. DSM ACC2737, deposited on Oct. 19,    2005-   b. 182-D1106-056, accession no. DSM ACC2738, deposited on Oct. 19,    2005-   c. 182-D1106-057, accession no. DSM ACC2739, deposited on Oct. 19,    2005-   d. 182-D1106-058, accession no. DSM ACC2740, deposited on Oct. 19,    2005-   e. 182-D1106-059, accession no. DSM ACC2741, deposited on Oct. 19,    2005-   f. 182-D1106-062, accession no. DSM ACC2742, deposited on Oct. 19,    2005,-   g. 182-D1106-067, accession no. DSM ACC2743, deposited on Oct. 19,    2005-   h. 182-D758-035, accession no. DSM ACC2745, deposited on Nov. 17,    2005-   i. 182-D758-036, accession no. DSM ACC2746, deposited on Nov. 17,    2005-   j. 182-D758-040, accession no. DSM ACC2747, deposited on Nov. 17,    2005-   k. 182-D1106-061, accession no. DSM ACC2748, deposited on Nov. 17,    2005-   l. 182-D1106-279, accession no. DSM ACC2808, deposited on Oct. 26,    2006-   m. 182-D1106-294, accession no. DSM ACC2809, deposited on Oct. 26,    2006,-   n. 182-D1106-362, accession no. DSM ACC2810, deposited on Oct. 26,    2006.

Preferred antibodies according to the invention are those produced byand obtainable from the above-described hybridomas; i.e. 37G11 in thecase of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in thecase of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in thecase of 182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in thecase of 182-D1106-067, 26B5 in the case of 182-D758-035, 26D12 in thecase of 182-D758-036, 28D10 in the case of 182-D758-040, 42E12 in thecase of 182-D1106-061, 125E1 in the case of 182-D1106-279, 163E12 in thecase of 182-D1106-294, and 175D10 in the case of 182-D1106-362; and thechimerized and humanized forms thereof.

Preferred chimerized antibodies and their sequences are shown in thefollowing table.

variable chimerized clone mAb Isotype region antibody heavy 43A11182-D1106-062 IgG2a SEQ ID SEQ ID chain NO: 29 NO: 14 163E12182-D1106-294 IgG3 SEQ ID SEQ ID NO: 30 NO: 15 125E1 182-D1106-279 IgG2aSEQ ID SEQ ID NO: 31 NO: 16 166E2 182-D1106-308 IgG3 SEQ ID SEQ ID NO:33 NO: 18 175D10 182-D1106-362 IgG1 SEQ ID SEQ ID NO: 32 NO: 17 45C1182-D758-187 IgG2a SEQ ID SEQ ID NO: 34 NO: 19 light 43A11 182-D1106-062IgK SEQ ID SEQ ID chain NO: 36 NO: 21 163E12 182-D1106-294 IgK SEQ IDSEQ ID NO: 35 NO: 20 125E1 182-D1106-279 IgK SEQ ID SEQ ID NO: 37 NO: 22166E2 182-D1106-308 IgK SEQ ID SEQ ID NO: 40 NO: 25 175D10 182-D1106-362IgK SEQ ID SEQ ID NO: 39 NO: 24 45C1 182-D758-187 IgK SEQ ID SEQ ID NO:38 NO: 23 45C1 182-D758-187 IgK SEQ ID SEQ ID NO: 41 NO: 26 45C1182-D758-187 IgK SEQ ID SEQ ID NO: 42 NO: 27 45C1 182-D758-187 IgK SEQID SEQ ID NO: 43 NO: 28

In preferred embodiments, antibodies, in particular chimerised forms ofantibodies according to the invention include antibodies comprising aheavy chain constant region (CH) comprising an amino acid sequencederived from a human heavy chain constant region such as the amino acidsequence represented by SEQ ID NO: 13 or a fragment thereof. In furtherpreferred embodiments, antibodies, in particular chimerised forms ofantibodies according to the invention include antibodies comprising alight chain constant region (CL) comprising an amino acid sequencederived from a human light chain constant region such as the amino acidsequence represented by SEQ ID NO: 12 or a fragment thereof. In aparticular preferred embodiment, antibodies, in particular chimerisedforms of antibodies according to the invention include antibodies whichcomprise a CH comprising an amino acid sequence derived from a human CHsuch as the amino acid sequence represented by SEQ ID NO: 13 or afragment thereof and which comprise a CL comprising an amino acidsequence derived from a human CL such as the amino acid sequencerepresented by SEQ ID NO: 12 or a fragment thereof.

In one embodiment, an antibody having the ability of binding to CLDN18.2is a chimeric mouse/human IgG1 monoclonal antibody comprising kappa,murine variable light chain, human kappa light chain constant regionallotype Km(3), murine heavy chain variable region, human IgG1 constantregion, allotype G1m(3).

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18,19, and a fragment thereof and/or comprising a light chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:20, 21, 22, 23, 24, 25, 26, 27, 28, and a fragment thereof.

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a combination of heavy chains and light chainsselected from the following possibilities (i) to (ix):

(i) the heavy chain comprises an amino acid sequence represented by SEQID NO: 14 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 21 or a fragment thereof,

(ii) the heavy chain comprises an amino acid sequence represented by SEQID NO: 15 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 20 or a fragment thereof,

(iii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 16 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 22 or a fragment thereof,

(iv) the heavy chain comprises an amino acid sequence represented by SEQID NO: 18 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 25 or a fragment thereof,

(v) the heavy chain comprises an amino acid sequence represented by SEQID NO: 17 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 24 or a fragment thereof,

(vi) the heavy chain comprises an amino acid sequence represented by SEQID NO: 19 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 23 or a fragment thereof,

(vii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 19 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 26 or a fragment thereof,

(viii) the heavy chain comprises an amino acid sequence represented bySEQ ID NO: 19 or a fragment thereof and the light chain comprises anamino acid sequence represented by SEQ ID NO: 27 or a fragment thereof,and

(ix) the heavy chain comprises an amino acid sequence represented by SEQID NO: 19 or a fragment thereof and the light chain comprises an aminoacid sequence represented by SEQ ID NO: 28 or a fragment thereof.

“Fragment” or “fragment of an amino acid sequence” as used above relatesto a part of an antibody sequence, i.e. a sequence which represents theantibody sequence shortened at the N- and/or C-terminus, which when itreplaces said antibody sequence in an antibody retains binding of saidantibody to CLDN18.2 and preferably functions of said antibody asdescribed herein, e.g. CDC mediated lysis or ADCC mediated lysis.Preferably, a fragment of an amino acid sequence comprises at least 80%,preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acidresidues from said amino acid sequence. A fragment of an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 preferably relates tosaid sequence wherein 17, 18, 19, 20, 21, 22 or 23 amino acids at theN-terminus are removed.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a heavy chain variable region (VH) comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:29, 30, 31, 32, 33, 34, and a fragment thereof.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a light chain variable region (VL) comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 35,36, 37, 38, 39, 40, 41, 42, 43, and a fragment thereof.

In certain preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 comprises a combination of heavy chain variableregion (VH) and light chain variable region (VL) selected from thefollowing possibilities (i) to (ix):

(i) the VH comprises an amino acid sequence represented by SEQ ID NO: 29or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 36 or a fragment thereof,

(ii) the VH comprises an amino acid sequence represented by SEQ ID NO:30 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 35 or a fragment thereof,

(iii) the VH comprises an amino acid sequence represented by SEQ ID NO:31 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 37 or a fragment thereof,

(iv) the VH comprises an amino acid sequence represented by SEQ ID NO:33 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 40 or a fragment thereof,

(v) the VH comprises an amino acid sequence represented by SEQ ID NO: 32or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 39 or a fragment thereof,

(vi) the VH comprises an amino acid sequence represented by SEQ ID NO:34 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 38 or a fragment thereof,

(vii) the VH comprises an amino acid sequence represented by SEQ ID NO:34 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 41 or a fragment thereof,

(viii) the VH comprises an amino acid sequence represented by SEQ ID NO:34 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 42 or a fragment thereof,

(ix) the VH comprises an amino acid sequence represented by SEQ ID NO:34 or a fragment thereof and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 43 or a fragment thereof.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a VH comprising a set of complementarity-determiningregions CDR1, CDR2 and CDR3 selected from the following embodiments (i)to (vi):

(i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14,

(ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77 ofSEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15,

(iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 ofSEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16,

(iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 ofSEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17,

(v) CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, and

(vi) CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 ofSEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a VL comprising a set of complementarity-determiningregions CDR1, CDR2 and CDR3 selected from the following embodiments (i)to (ix):

(i) CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20,

(ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 ofSEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO: 21,

(iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 ofSEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 ofSEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23,

(v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQID NO: 24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 ofSEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO: 25,

(vii) CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 ofSEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 ofSEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO: 27, and

(ix) CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 ofSEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO: 28.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a combination of VH and VL each comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 selected fromthe following embodiments (i) to (ix):

(i) VH: CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 ofSEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, VL: CDR1:positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO:21, CDR3: positions 110-118 of SEQ ID NO: 21,

(ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions 70-77of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO: 15, VL: CDR1:positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 of SEQ ID NO:20, CDR3: positions 115-123 of SEQ ID NO: 20,

(iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1:positions 47-52 of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO:22, CDR3: positions 109-117 of SEQ ID NO: 22,

(iv) VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, VL: CDR1:positions 47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO:25, CDR3: positions 115-122 of SEQ ID NO: 25,

(v) VH: CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions 70-77 ofSEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO: 17, VL: CDR1:positions 47-58 of SEQ ID NO: 24, CDR2: positions 76-78 of SEQ ID NO:24, CDR3: positions 115-123 of SEQ ID NO: 24,

(vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1:positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 of SEQ ID NO:23, CDR3: positions 115-123 of SEQ ID NO: 23,

(vii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1:positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO:26, CDR3: positions 115-123 of SEQ ID NO: 26,

(viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1:positions 47-58 of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO:27, CDR3: positions 115-123 of SEQ ID NO: 27, and

(ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1:positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 of SEQ ID NO:28, CDR3: positions 109-117 of SEQ ID NO: 28.

In further preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 preferably comprises one or more of thecomplementarity-determining regions (CDRs), preferably at least the CDR3variable region, of the heavy chain variable region (VH) and/or of thelight chain variable region (VL) of a monoclonal antibody againstCLDN18.2, preferably of a monoclonal antibody against CLDN18.2 describedherein, and preferably comprises one or more of thecomplementarity-determining regions (CDRs), preferably at least the CDR3variable region, of the heavy chain variable regions (VH) and/or lightchain variable regions (VL) described herein. In one embodiment said oneor more of the complementarity-determining regions (CDRs) are selectedfrom a set of complementarity-determining regions CDR1, CDR2 and CDR3described herein. In a particularly preferred embodiment, an antibodyhaving the ability of binding to CLDN18.2 preferably comprises thecomplementarity-determining regions CDR1, CDR2 and CDR3 of the heavychain variable region (VH) and/or of the light chain variable region(VL) of a monoclonal antibody against CLDN18.2, preferably of amonoclonal antibody against CLDN18.2 described herein, and preferablycomprises the complementarity-determining regions CDR1, CDR2 and CDR3 ofthe heavy chain variable regions (VH) and/or light chain variableregions (VL) described herein.

In one embodiment an antibody comprising one or more CDRs, a set of CDRsor a combination of sets of CDRs as described herein comprises said CDRstogether with their intervening framework regions. Preferably, theportion will also include at least about 50% of either or both of thefirst and fourth framework regions, the 50% being the C-terminal 50% ofthe first framework region and the N-terminal 50% of the fourthframework region. Construction of antibodies made by recombinant DNAtechniques may result in the introduction of residues N- or C-terminalto the variable regions encoded by linkers introduced to facilitatecloning or other manipulation steps, including the introduction oflinkers to join variable regions of the invention to further proteinsequences including immunoglobulin heavy chains, other variable domains(for example in the production of diabodies) or protein labels.

In one embodiment an antibody comprising one or more CDRs, a set of CDRsor a combination of sets of CDRs as described herein comprises said CDRsin a human antibody framework.

Reference herein to an antibody comprising with respect to the heavychain thereof a particular chain, or a particular region or sequencepreferably relates to the situation wherein all heavy chains of saidantibody comprise said particular chain, region or sequence. Thisapplies correspondingly to the light chain of an antibody.

The term “nucleic acid”, as used herein, is intended to include DNA andRNA. A nucleic acid may be single-stranded or double-stranded, butpreferably is double-stranded DNA.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.

The teaching given herein with respect to specific amino acid sequences,e.g. those shown in the sequence listing, is to be construed so as toalso relate to variants of said specific sequences resulting insequences which are functionally equivalent to said specific sequences,e.g. amino acid sequences exhibiting properties identical or similar tothose of the specific amino acid sequences. One important property is toretain binding of an antibody to its target or to sustain effectorfunctions of an antibody. Preferably, a sequence which is a variant withrespect to a specific sequence, when it replaces the specific sequencein an antibody retains binding of said antibody to CLDN18.2 andpreferably functions of said antibody as described herein, e.g. CDCmediated lysis or ADCC mediated lysis.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR, hypervariable and variable regions can bemodified without losing the ability to bind CLDN18.2. For example, CDRregions will be either identical or highly homologous to the regions ofantibodies specified herein. By “highly homologous” it is contemplatedthat from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2substitutions may be made in the CDRs. In addition, the hypervariableand variable regions may be modified so that they show substantialhomology with the regions of antibodies specifically disclosed herein.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants. Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

The term “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably heavy and/or light chaintransgenes, or transchromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is preferably capableof expressing the transgenes. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humananti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and/orcells expressing CLDN18.2. The human heavy chain transgene can beintegrated into the chromosomal DNA of the mouse, as is the case fortransgenic mice, e.g., HuMAb mice, such as HCo7 or HCo12 mice, or thehuman heavy chain transgene can be maintained extrachromosomally, as isthe case for transchromosomal (e.g., KM) mice as described in WO02/43478. Such transgenic and transchromosomal mice may be capable ofproducing multiple isotypes of human monoclonal antibodies to CLDN18.2(e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination andisotype switching.

“Reduce”, “decrease” or “inhibit” as used herein means an overalldecrease or the ability to cause an overall decrease, preferably of 5%or greater, 10% or greater, 20% or greater, more preferably of 50% orgreater, and most preferably of 75% or greater, in the level, e.g. inthe level of expression or in the level of proliferation of cells.

Terms such as “increase” or “enhance” preferably relate to an increaseor enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%, at least 200%, at least 500%, at least 1000%, at least10000% or even more.

Mechanisms of mAb Action

Although the following provides considerations regarding the mechanismunderlying the therapeutic efficacy of antibodies of the invention it isnot to be considered as limiting to the invention in any way.

The antibodies described herein preferably interact with components ofthe immune system, preferably through ADCC or CDC. Antibodies describedherein can also be used to target payloads (e.g., radioisotopes, drugsor toxins) to directly kill tumor cells or can be used synergisticallywith traditional chemotherapeutic agents, attacking tumors throughcomplementary mechanisms of action that may include anti-tumor immuneresponses that may have been compromised owing to a chemotherapeutic'scytotoxic side effects on T lymphocytes. However, antibodies describedherein may also exert an effect simply by binding to CLDN18.2 on thecell surface, thus, e.g. blocking proliferation of the cells.

Antibody-Dependent Cell-Mediated Cytotoxicity

ADCC describes the cell-killing ability of effector cells as describedherein, in particular lymphocytes, which preferably requires the targetcell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cellsand the antibody Fc domains engage Fc receptors (FcR) on the surface ofimmune effector cells. Several families of Fc receptors have beenidentified, and specific cell populations characteristically expressdefined Fc receptors. ADCC can be viewed as a mechanism to directlyinduce a variable degree of immediate tumor destruction that leads toantigen presentation and the induction of tumor-directed T-cellresponses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and host-derived antibody responses.

Complement-Dependent Cytotoxicity

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple C1q binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (C1q is one ofthree subcomponents of complement C1). Preferably these uncloaked C1qbinding sites convert the previously low-affinity C1q-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell.

Antibodies described herein can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature 256: 495 (1975). Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibodies can be employed, e.g., viral or oncogenic transformation ofB-lymphocytes or phage display techniques using libraries of antibodygenes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodies can begenerated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice known as HuMAb mice and KM mice,respectively, and are collectively referred to herein as “transgenicmice.” The production of human antibodies in such transgenic mice can beperformed as described in detail for CD20 in WO2004 035607

Yet another strategy for generating monoclonal antibodies is to directlyisolate genes encoding antibodies from lymphocytes producing antibodiesof defined specificity e.g. see Babcock et al., 1996; A novel strategyfor generating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined specificities. For details ofrecombinant antibody engineering see also Welschof and Kraus,Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and BennyK. C. Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies, mice can be immunized with carrier-conjugatedpeptides derived from the antigen sequence, i.e. the sequence againstwhich the antibodies are to be directed, an enriched preparation ofrecombinantly expressed antigen or fragments thereof and/or cellsexpressing the antigen, as described. Alternatively, mice can beimmunized with DNA encoding the antigen or fragments thereof. In theevent that immunizations using a purified or enriched preparation of theantigen do not result in antibodies, mice can also be immunized withcells expressing the antigen, e.g., a cell line, to promote immuneresponses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of immunoglobulin canbe used for fusions. Mice can be boosted intraperitonealy orintravenously with antigen expressing cells 3 days before sacrifice andremoval of the spleen to increase the rate of specific antibodysecreting hybridomas.

To generate hybridomas producing monoclonal antibodies, splenocytes andlymph node cells from immunized mice can be isolated and fused to anappropriate immortalized cell line, such as a mouse myeloma cell line.The resulting hybridomas can then be screened for the production ofantigen-specific antibodies. Individual wells can then be screened byELISA for antibody secreting hybridomas. By Immunofluorescence and FACSanalysis using antigen expressing cells, antibodies with specificity forthe antigen can be identified. The antibody secreting hybridomas can bereplated, screened again, and if still positive for monoclonalantibodies can be subcloned by limiting dilution. The stable subclonescan then be cultured in vitro to generate antibody in tissue culturemedium for characterization.

Antibodies also can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as are well known in the art (Morrison, S. (1985)Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. Afterintroduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Chimerization

Murine monoclonal antibodies can be used as therapeutic antibodies inhumans when labeled with toxins or radioactive isotopes. Nonlabeledmurine antibodies are highly immunogenic in man when repetitivelyapplied leading to reduction of the therapeutic effect. The mainimmunogenicity is mediated by the heavy chain constant regions. Theimmunogenicity of murine antibodies in man can be reduced or completelyavoided if respective antibodies are chimerized or humanized. Chimericantibodies are antibodies, the different portions of which are derivedfrom different animal species, such as those having a variable regionderived from a murine antibody and a human immunoglobulin constantregion. Chimerisation of antibodies is achieved by joining of thevariable regions of the murine antibody heavy and light chain with theconstant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

Humanization

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A 86: 10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences. These germline sequences will differfrom mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region.

The ability of antibodies to bind an antigen can be determined usingstandard binding assays (e.g., ELISA, Western Blot, Immunofluorescenceand flow cytometric analysis).

To purify antibodies, selected hybridomas can be grown in two-literspinner-flasks for monoclonal antibody purification. Alternatively,antibodies can be produced in dialysis based bioreactors. Supernatantscan be filtered and, if necessary, concentrated before affinitychromatography with protein G-sepharose or protein A-sepharose. ElutedIgG can be checked by gel electrophoresis and high performance liquidchromatography to ensure purity. The buffer solution can be exchangedinto PBS, and the concentration can be determined by OD280 using 1.43extinction coefficient. The monoclonal antibodies can be aliquoted andstored at −80° C.

To determine if the selected monoclonal antibodies bind to uniqueepitopes, site-directed or multi-site directed mutagenesis can be used.

To determine the isotype of antibodies, isotype ELISAs with variouscommercial kits (e.g. Zymed, Roche Diagnostics) can be performed. Wellsof microtiter plates can be coated with anti-mouse Ig. After blocking,the plates are reacted with monoclonal antibodies or purified isotypecontrols, at ambient temperature for two hours. The wells can then bereacted with either mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouseIgM-specific peroxidase-conjugated probes. After washing, the plates canbe developed with ABTS substrate (1 mg/ml) and analyzed at OD of405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody IsotypingKit (Roche, Cat. No. 1493027) may be used as described by themanufacturer.

In order to demonstrate presence of antibodies in sera of immunized miceor binding of monoclonal antibodies to living cells expressing antigen,flow cytometry can be used. Cell lines expressing naturally or aftertransfection antigen and negative controls lacking antigen expression(grown under standard growth conditions) can be mixed with variousconcentrations of monoclonal antibodies in hybridoma supernatants or inPBS containing 1% FBS, and can be incubated at 4° C. for 30 min. Afterwashing, the APC- or Alexa647-labeled anti IgG antibody can bind toantigen-bound monoclonal antibody under the same conditions as theprimary antibody staining. The samples can be analyzed by flow cytometrywith a FACS instrument using light and side scatter properties to gateon single, living cells. In order to distinguish antigen-specificmonoclonal antibodies from non-specific binders in a single measurement,the method of co-transfection can be employed. Cells transientlytransfected with plasmids encoding antigen and a fluorescent marker canbe stained as described above. Transfected cells can be detected in adifferent fluorescence channel than antibody-stained cells. As themajority of transfected cells express both transgenes, antigen-specificmonoclonal antibodies bind preferentially to fluorescence markerexpressing cells, whereas non-specific antibodies bind in a comparableratio to non-transfected cells. An alternative assay using fluorescencemicroscopy may be used in addition to or instead of the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy.

In order to demonstrate presence of antibodies in sera of immunized miceor binding of monoclonal antibodies to living cells expressing antigen,immunofluorescence microscopy analysis can be used. For example, celllines expressing either spontaneously or after transfection antigen andnegative controls lacking antigen expression are grown in chamber slidesunder standard growth conditions in DMEM/F12 medium, supplemented with10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and100 μg/ml streptomycin. Cells can then be fixed with methanol orparaformaldehyde or left untreated. Cells can then be reacted withmonoclonal antibodies against the antigen for 30 min. at 25° C. Afterwashing, cells can be reacted with an Alexa555-labelled anti-mouse IgGsecondary antibody (Molecular Probes) under the same conditions. Cellscan then be examined by fluorescence microscopy.

Cell extracts from cells expressing antigen and appropriate negativecontrols can be prepared and subjected to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked, andprobed with the monoclonal antibodies to be tested. IgG binding can bedetected using anti-mouse IgG peroxidase and developed with ECLsubstrate.

Antibodies can be further tested for reactivity with antigen byImmunohistochemistry in a manner well known to the skilled person, e.g.using paraformaldehyde or acetone fixed cryosections or paraffinembedded tissue sections fixed with paraformaldehyde from non-cancertissue or cancer tissue samples obtained from patients during routinesurgical procedures or from mice carrying xenografted tumors inoculatedwith cell lines expressing spontaneously or after transfection antigen.For immunostaining, antibodies reactive to antigen can be incubatedfollowed by horseradish-peroxidase conjugated goat anti-mouse or goatanti-rabbit antibodies (DAKO) according to the vendors instructions.

Antibodies can be tested for their ability to mediate phagocytosis andkilling of cells expressing CLDN18.2. The testing of monoclonal antibodyactivity in vitro will provide an initial screening prior to testing invivo models.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC):

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes,mononuclear cells or other effector cells, from healthy donors can bepurified by Ficoll Hypaque density centrifugation, followed by lysis ofcontaminating erythrocytes. Washed effector cells can be suspended inRPMI supplemented with 10% heat-inactivated fetal calf serum or,alternatively with 5% heat-inactivated human serum and mixed with ⁵¹Crlabeled target cells expressing CLDN18.2, at various ratios of effectorcells to target cells. Alternatively, the target cells may be labeledwith a fluorescence enhancing ligand (BATDA). A highly fluorescentchelate of Europium with the enhancing ligand which is released fromdead cells can be measured by a fluorometer. Another alternativetechnique may utilize the transfection of target cells with luciferase.Added lucifer yellow may then be oxidated by viable cells only. Purifiedanti-CLDN18.2 IgGs can then be added at various concentrations.Irrelevant human IgG can be used as negative control. Assays can becarried out for 4 to 20 hours at 37° C. depending on the effector celltype used. Samples can be assayed for cytolysis by measuring ⁵¹Crrelease or the presence of the EuTDA chelate in the culture supernatant.Alternatively, luminescence resulting from the oxidation of luciferyellow can be a measure of viable cells. Anti-CLDN18.2 monoclonalantibodies can also be tested in various combinations to determinewhether cytolysis is enhanced with multiple monoclonal antibodies.

Complement Dependent Cytotoxicity (CDC):

Monoclonal anti-CLDN18.2 antibodies can be tested for their ability tomediate CDC using a variety of known techniques. For example, serum forcomplement can be obtained from blood in a manner known to the skilledperson. To determine the CDC activity of mAbs, different methods can beused. ⁵¹Cr release can for example be measured or elevated membranepermeability can be assessed using a propidium iodide (PI) exclusionassay. Briefly, target cells can be washed and 5×10⁵/ml can be incubatedwith various concentrations of mAb for 10-30 min. at room temperature orat 37° C. Serum or plasma can then be added to a final concentration of20% (v/v) and the cells incubated at 37° C. for 20-30 min. All cellsfrom each sample can be added to the PI solution in a FACS tube. Themixture can then be analyzed immediately by flow cytometry analysisusing FACSArray.

In an alternative assay, induction of CDC can be determined on adherentcells. In one embodiment of this assay, cells are seeded 24 h before theassay with a density of 3×10⁴/well in tissue-culture flat-bottommicrotiter plates. The next day growth medium is removed and the cellsare incubated in triplicates with antibodies. Control cells areincubated with growth medium or growth medium containing 0.2% saponinfor the determination of background lysis and maximal lysis,respectively. After incubation for 20 min. at room temperaturesupernatant is removed and 20% (v/v) human plasma or serum in DMEM(prewarmed to 37° C.) is added to the cells and incubated for another 20min. at 37° C. All cells from each sample are added to propidium iodidesolution (10 μg/ml). Then, supernatants are replaced by PBS containing2.5 μg/ml ethidium bromide and fluorescence emission upon excitation at520 nm is measured at 600 nm using a Tecan Safire. The percentagespecific lysis is calculated as follows: % specific lysis=(fluorescencesample-fluorescence background)/(fluorescence maximal lysis-fluorescencebackground)×100.

Induction of Apoptosis and Inhibition of Cell Proliferation byMonoclonal Antibodies:

To test for the ability to initiate apoptosis, monoclonal anti-CLDN18.2antibodies can, for example, be incubated with CLDN18.2 positive tumorcells, e.g., SNU-16, DAN-G, KATO-III or CLDN18.2 transfected tumor cellsat 37° C. for about 20 hours. The cells can be harvested, washed inAnnexin-V binding buffer (BD biosciences), and incubated with Annexin Vconjugated with FITC or APC (BD biosciences) for 15 min. in the dark.All cells from each sample can be added to PI solution (10 μg/ml in PBS)in a FACS tube and assessed immediately by flow cytometry (as above).Alternatively, a general inhibition of cell-proliferation by monoclonalantibodies can be detected with commercially available kits. The DELFIACell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopicimmunoassay based on the measurement of 5-bromo-2′-deoxyuridine (BrdU)incorporation during DNA synthesis of proliferating cells inmicroplates. Incorporated BrdU is detected using europium labelledmonoclonal antibody. To allow antibody detection, cells are fixed andDNA denatured using Fix solution. Unbound antibody is washed away andDELFIA inducer is added to dissociate europium ions from the labelledantibody into solution, where they form highly fluorescent chelates withcomponents of the DELFIA Inducer. The fluorescence measured—utilizingtime-resolved fluorometry in the detection—is proportional to the DNAsynthesis in the cell of each well.

Preclinical Studies

Monoclonal antibodies which bind to CLDN18.2 also can be tested in an invivo model (e.g. in immune deficient mice carrying xenografted tumorsinoculated with cell lines expressing CLDN18.2, e.g. DAN-G, SNU-16, orKATO-III, or after transfection, e.g. HEK293) to determine theirefficacy in controlling growth of CLDN18.2-expressing tumor cells.

In vivo studies after xenografting CLDN18.2 expressing tumor cells intoimmunocompromised mice or other animals can be performed usingantibodies described herein. Antibodies can be administered to tumorfree mice followed by injection of tumor cells to measure the effects ofthe antibodies to prevent formation of tumors or tumor-related symptoms.Antibodies can be administered to tumor-bearing mice to determine thetherapeutic efficacy of respective antibodies to reduce tumor growth,metastasis or tumor related symptoms. Antibody application can becombined with application of other substances as cystostatic drugs,growth factor inhibitors, cell cycle blockers, angiogenesis inhibitorsor other antibodies to determine synergistic efficacy and potentialtoxicity of combinations. To analyze toxic side effects mediated byantibodies animals can be inoculated with antibodies or control reagentsand thoroughly investigated for symptoms possibly related toCLDN18.2-antibody therapy. Possible side effects of in vivo applicationof CLDN18.2 antibodies particularly include toxicity at CLDN18.2expressing tissues including stomach.

Antibodies recognizing CLDN18.2 in human and in other species, e.g.mice, are particularly useful to predict potential side effects mediatedby application of monoclonal CLDN18.2-antibodies in humans.

Mapping of epitopes recognized by antibodies can be performed asdescribed in detail in “Epitope Mapping Protocols (Methods in MolecularBiology) by Glenn E. Morris ISBN-089603-375-9 and in “Epitope Mapping: APractical Approach” Practical Approach Series, 248 by Olwyn M. R.Westwood, Frank C. Hay.

The compounds and agents described herein may be administered in theform of any suitable pharmaceutical composition.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. A pharmaceuticalcomposition may e.g. be in the form of a solution or suspension.

A pharmaceutical composition may comprise salts, buffer substances,preservatives, carriers, diluents and/or excipients all of which arepreferably pharmaceutically acceptable. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinteract with the action of the active component of the pharmaceuticalcomposition.

Salts which are not pharmaceutically acceptable may used for preparingpharmaceutically acceptable salts and are included in the invention.Pharmaceutically acceptable salts of this kind comprise in a nonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in a pharmaceutical compositioninclude acetic acid in a salt, citric acid in a salt, boric acid in asalt and phosphoric acid in a salt.

Suitable preservatives for use in a pharmaceutical composition includebenzalkonium chloride, chlorobutanol, paraben and thimerosal.

An injectable formulation may comprise a pharmaceutically acceptableexcipient such as Ringer Lactate.

The term “carrier” refers to an organic or inorganic component, of anatural or synthetic nature, in which the active component is combinedin order to facilitate, enhance or enable application. According to theinvention, the term “carrier” also includes one or more compatible solidor liquid fillers, diluents or encapsulating substances, which aresuitable for administration to a patient.

Possible carrier substances for parenteral administration are e.g.sterile water, Ringer, Ringer lactate, sterile sodium chloride solution,polyalkylene glycols, hydrogenated naphthalenes and, in particular,biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition andwhich are not active ingredients such as, e.g., carriers, binders,lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The agents and compositions described herein may be administered via anyconventional route, such as by parenteral administration including byinjection or infusion. Administration is preferably parenterally, e.g.intravenously, intraarterially, subcutaneously, intradermally orintramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The agents and compositions described herein are administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on various of such parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The agents and compositions described herein can be administered topatients, e.g., in vivo, to treat or prevent a variety of disorders suchas those described herein. Preferred patients include human patientshaving disorders that can be corrected or ameliorated by administeringthe agents and compositions described herein. This includes disordersinvolving cells characterized by an altered expression pattern ofCLDN18.2.

For example, in one embodiment, antibodies described herein can be usedto treat a patient with a cancer disease, e.g., a cancer disease such asdescribed herein characterized by the presence of cancer cellsexpressing CLDN18.2.

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to prevent a disease described herein.

The present invention is further illustrated by the following exampleswhich are not be construed as limiting the scope of the invention.

EXAMPLES Example 1: CLDN18.2 Expression of Human Gastric Cancer CellLines is Stabilized by In Vitro Treatment with Chemotherapeutic Agents

KatoIII cells, a human gastric tumor cell line, was cultivated in RPMI1640 medium (Invitrogen) containing 20% FCS (Perbio) and 2 mM Glutamax(Invitrogen) at 37° C. and 5% CO₂, with or without cytostatic compounds.Epirubicin (Pfizer) was tested at a concentration of 10 or 100 ng/ml,5-FU (Neofluor from NeoCorp AG) was tested at a concentration of 10 or100 ng/ml, and oxaliplatin (Hospira) was tested at a concentration of 50or 500 ng/ml. A combination of all 3 compounds (EOF; epirubicin 10ng/ml, oxaliplatin 500 ng/ml, 5-FU 10 ng/ml) was also used. 8×10⁵KatoIII cells were cultivated for 96 hours without medium change or for72 hours followed by 24 hours cultivation in standard medium to releasecells from cell cycle arrest in a 6-well tissue culture plate at 37° C.,5% CO₂. Cells were harvested with EDTA/trypsin, washed and analysed.

For extracellular detection of CLDN18.2 cells were stained with themonoclonal anti-CLDN18.2 antibody IMAB362 (Ganymed) or an isotyp-matchedcontrol antibody (Ganymed). As secondary reagent goat-anti-hulgG-APCfrom Dianova was used.

Cell cycle stages were determined based on measurement of cellular DNAcontent. This allows one to discriminate between cells in the G1-, S- orG2-phase of the cell cycle. In the S-phase DNA duplication occurswhereas in the G2-phase cells grow and prepare for mitosis. Cell cycleanalysis was done using the CycleTEST PLUS DNA Reagent Kit from BDBiosciences following the manufacturer's protocol. Flow cytometryacquisition and analysis were performed by using BD FACS CantoII (BDBiosciences) and FlowJo (Tree Star) software.

The columns in FIGS. 1a and b show the respective percentage of cells inthe G1-, S- or G2-phase of the cell cycle. Medium cultivated KatoIIIcells show a cell cycle arrest predominantly in the G1-phase. Cellstreated with 5-FU are blocked predominantly in the 5-phase. Epirubicin-or EOF-treated KatoIII cells show a cell cycle arrest predominantly inthe G2-phase. Oxaliplatin treated KatoIII cells show enrichment of cellspredominantly in the G1- and G2-phases. As can be seen in FIG. 1c , acell cycle arrest in the S-phase or G2-phase results in stabilization orupregulation of CLDN18.2. As soon as cells are released from any phaseof the cell cycle (FIG. 1b ) the expression of CLDN18.2 on the cellsurface of KatoIII cells is upregulated (FIG. 1d ).

NUGC-4 and KATO III cells were treated with 5-FU+OX (10 ng/ml 5-FU and500 ng/ml oxaliplatin), EOF (10 ng/ml epirubicin, 500 ng/ml oxaliplatinand 10 ng/ml 5-FU) or FLO (10 ng/ml 5-FU, 50 ng/ml folinic acid and 500ng/ml oxaliplatin) for 96 hours. RNA of chemotherapy pretreated NUGC-4and KATO III cells was isolated and converted to cDNA. CLDN18.2transcript level was analysed in quantitative real-time PCR. Results areshown in FIG. 2a as relative expression in comparison to the transcriptlevel of the housekeeping gene HPRT. FIG. 2b shows a Western blot ofCLDN18.2 and actin loading control of untreated and treated NUGC-4cells. The intensity of the luminescence signal is shown in relation toactin in percent.

Pretreatment of NUGC-4 and KATO III cells with EOF, FLO as well as5-FU+OX combination chemotherapies results in increased RNA and proteinlevels of CLDN18.2 as shown by quantitative real-time PCR (FIG. 2a ) andWestern blot (FIG. 2b ).

IMAB362 binding on NUGC-4 and KATO III gastric cancer cells treated withEOF (10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml 5-FU) orFLO (10 ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/ml oxaliplatin) for96 hours by flow cytometry was analysed. The amount of CLDN18.2 proteintargetable by IMAB362 on the surface of gastric cancer cell lines isincreased as shown in FIG. 2c . This effect was most prominent in cellspretreated with EOF or FLO.

KatoIII cells were pretreated for 4 days with Irinotecan or Docetaxeland analysed for CLDN18.2 expression and cell cycle arrest. Treatment ofcells with Irinotecan resulted in a dose dependent inhibition of cellgrowth and a cell cycle arrest in the S/G2-phase (FIG. 3). Treatment ofcells with Docetaxel resulted in a dose dependent inhibition of cellgrowth and a cell cycle arrest in the G2-phase (FIG. 3).

Example 2: Pretreatment of Human Gastric Cancer Cells withChemotherapeutics Results in Higher Efficiency of IMAB362-Mediated ADCC

IMAB362-mediated ADCC was investigated using NUGC-4 gastric cancer cellsas target, which were either pretreated with 10 ng/ml 5-FU and 500 ng/mloxaliplatin (5-FU+OX), 10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10ng/ml 5-FU (EOF) or 10 ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/mloxaliplatin (FLO) for 96 hours (effector:target ratio 40:1) oruntreated. EC₅₀ values were obtained from 7 healthy donors for untreatedand EOF, FLO or 5-FU+OX pretreated NUGC-4 cells.

As shown in FIG. 4a , dose/response curves on pretreated cells shiftedupwards and to the left compared to untreated target cells. Thisresulted in a higher maximal lysis and in a decrease of the EC₅₀ valuesto one third of untreated cells (FIG. 4b ).

Peripheral blood mononuclear cells (PBMCs) including NK cells,monocytes, mononuclear cells or other effector cells from healthy humandonors were purified by Ficoll Hypaque density centrifugation. Washedeffector cells were seeded in X-Vivo medium. KatoIII cells which expressCLDN18.2 endogenously and are of gastric origin were used as targetcells in this setting. Target cells stably expressed luciferase, luciferyellow, which is oxidized by viable cells only. Purified anti-CLDN18.2antibody IMAB362 was added at various concentrations and as an isotypecontrol antibody an irrelevant chim huIgG1 antibody was used. Sampleswere assayed for cytolysis by measuring luminescence resulting from theoxidation of lucifer yellow which is a value for the amount of viablecells left after IMAB362 induced cytotoxicity. KatoIII pretreated for 3days with Irinotecan (1000 ng/ml), Docetaxel (5 ng/ml) or Cisplatin(2000 ng/ml) were compared to untreated medium cultivated target cellsand IMAB362 induced ADCC was quantified.

KatoIII cells pretreated for 3 days with Irinotecan, Docetaxel orCisplatin exhibited a lower level of viable cells compared to mediumcultivated target cells (FIG. 5a ) and claudin18.2 expression in cellspretreated with Irinotecan, Docetaxel or Cisplatin was increasedcompared to medium cultivated cells (FIG. 5b ).

Furthermore, pretreatment of KatoIII cells with Irinotecan, Docetaxel orCisplatin augmented the potency of IMAB362 to induce ADCC (FIG. 5c, d ).

Example 3: Chemotherapy Results in Higher Efficiency of IMAB362-InducedCDC

Effects of chemotherapeutic agents on IMAB362-induced CDC were analyzedby pretreating KATO III gastric cancer cells with 10 ng/ml 5-FU and 500ng/ml oxaliplatin (5-FU+OX) for 48 hours. Representative dose responsecurves of IMAB362-induced CDC using chemotherapeutic pretreated KATO IIIcells are shown in FIG. 6. Pretreatment of tumor cells for 48 hoursaugmented the potency of IMAB362 to induce CDC, resulting in highermaximal cell lysis of pretreated tumor cells compared to untreatedcells.

Example 4: Capability of Immune Effector Cells to ExecuteIMAB362-Mediated ADCC is not Compromised by Treatment withChemotherapeutics

Chemotherapeutic agents used in EOF or FLO regimen are highly potent ininhibition of target cell proliferation. To investigate adverse effectsof chemotherapy on effector cells, PBMCs from healthy donors weretreated with 10 ng/ml epirubicin, 500 ng/ml oxaliplatin and 10 ng/ml5-FU (EOF) or 10 ng/ml 5-FU, 50 ng/ml folinic acid and 500 ng/mloxaliplatin (FLO) for 72 hours before application in ADCC assays. FIG.7a shows EC₅₀ values of 4 healthy donors and FIG. 7b showsrepresentative dose/response curves of IMAB362-induced ADCC using EOF orFLO pretreated effector cells. IMAB362-induced ADCC of NUGC-4 gastriccarcinoma cells is not compromised due to EOF or FLO chemotherapies.

Example 5: A Combination of ZA/IL-2 Treatment Results in OptimizedExpansion of Peripheral Blood Mononuclear Cell (PBMC) Cultures

The effect of ZA/IL-2 on proliferation of PBMC cultures was assessed invitro. PBMCs were harvested from healthy human donors and cultures weretreated with a single dose of ZA. IL-2 was added every 3-4 days.Specifically, PBMC derived from 3 different healthy human donors (#1,#2, #3) were cultured in RPMI medium (1×10⁶ cells/ml) for 14 days with 1μM ZA plus high (300 U/ml) or low (25 U/ml) doses of IL-2; cf. FIG. 8a .PBMCs of the same donors were cultured additionally in RPMI medium for14 days with 300 U/ml IL-2 plus ZA or without ZA; cf. FIG. 8b . Increasein cell numbers was determined by counting living cells on day 6, 8, 11and 14.

In medium supplied with a high dose of IL-2 about 2-5 fold more cellsexpanded, as compared to cultures supplied with a low IL-2 dose (FIG. 8a). Expansion of cells in medium without ZA was approximately 2 foldlower as compared to cells grown in medium with ZA (FIG. 8b ). Thesedata show the necessity to apply both ZA and IL-2 compounds incombination to ensure proper expansion of the cells.

Example 6: ZA/IL-2 Treatment Results in Expansion of High Amounts ofVγ9Vδ2 T-Cells in PBMC Cultures

PBMCs were cultivated for 14 days in RPMI medium supplemented with 300U/ml IL-2 and with or w/o 1 μM ZA. The percentage of Vγ9+Vδ2+ T cellswithin the CD3+ lymphocyte population (FIG. 9a ) and the percentage ofCD16+ cells within the CD3+Vγ9+Vδ2+ T cell population (FIG. 9b ) wasdetermined by multicolor FACS on day 0 and day 14. Results were scoredfor each donor in the scatter plot. FIG. 9c shows a scatter plotdisplaying the increase over time (enrichment) in the number ofCD3+Vγ9+Vδ2+ and CD3+CD16+Vγ9+Vδ2+ T cells within the lymphocytepopulation. The amount of cells seeded on day 0 and the amount of cellsharvested on day 14 were taken into account.

IL-2 addition in the PBMC cultures is required for survival and growthof lymphocytes. They efficiently expand in cultures supplied with 300U/ml IL-2. FACS analysis using Vγ9 and Vδ2 specific antibodies revealthat addition of ZA/IL-2 specifically induces the accumulation of Vγ9Vδ2T cells (FIG. 9a ). After 14 days, the CD3+ lymphocyte population cancomprise up to 80% of Vγ9Vδ2 T cells. A portion of Vγ9Vδ2 T cellsexpress CD16, whereas enrichment of these cells within the CD3+lymphocyte population is 10-700 fold, dependent on the donor (FIGS. 9band 9c ). Enrichment of the CD16+Vγ9+Vδ2+ T cells in the cultures is10-600 fold higher as compared to cultures grown without ZA (FIG. 9c ).We conclude that ZA/IL-2 treatment of PBMCs in vitro results in theup-regulation of the ADCC-mediating FcγIII receptor CD16 in asignificant proportion of γδ T cells.

Example 7: IL-2 Affects Expansion of Vγ9Vδ2 T Cells in a Dose-DependentManner

Addition of ZA in the cultures is the most important factor to inducedevelopment of Vγ9Vδ2 T cells. It is well known, that IL-2 is requiredfor growth and survival of T cells.

PBMCs were cultivated for 14 days in RPMI medium supplemented with 1 μMZA and increasing IL-2 concentrations. IL-2 was added on day 0 and day4. The enrichment of CD16+Vγ9+Vδ2+ T cells within the CD3+ lymphocytepopulation was determined by multicolor FACS staining on day 0 and day14. To compare the different donors, the amount of CD16+Vγ9+Vδ2+ T cellsharvested after cultivation with 600 U/ml IL-2 was set to 100%; cf. FIG.10, left. Furthermore, ADCC activity of the isolated cultures grown for14 days in increasing concentrations of IL-2 was tested; cf. FIG. 10,right.

We confirmed by dose response analysis that IL-2 also stimulates growthand survival of the Vγ9Vδ2 T cell subset. By adding low IL-2concentrations in the medium, a correlation was found between IL-2 doseand the percentage of CD16+Vγ9Vδ2 T cells within the CD3+ lymphocytepopulation (FIG. 10, left). ADCC activity of the cells grown in higherIL-2 concentrations (150-600U/ml) is improved compared to cells grown inlow IL-2 concentrations (FIG. 10, right).

Example 8: ZA Induces IPP Production in Monocytes and Cancer CellsStimulating Both the Expansion of Vγ9Vδ2 T Cells

Fresh PBMCs (Exp. #1) or 14 day ZA/IL-2 stimulated Vγ9Vδ2 T cellcultures (Exp. #2-5) were incubated either without monocytes(effector:monocyte ratio 1:0), with 0.2 fold (4:1) or 5 fold (ratio 1:4)the amount of monocytes ±1 μM ZA. The enrichment of Vγ9Vδ2 T cells inthe co-cultures after 14 days was determined by multicolor FACS, whereasthe expansion of the culture was considered in the calculation. Theenrichment factor of Vγ9Vδ2 T cells cultured with monocytes in a 1:4ratio was set to 100% for each experiment. The increase of monocytes inthe culture resulted in an enrichment of Vγ9Vδ2 T cells of more than 10fold. This effect was clearly ZA-dependent; cf. FIG. 11 a.

Furthermore, human stomach cancer cells (NUGC-4-luciferase) and murinestomach cancer cells (CLS103—calcein stained) were pretreated with orw/o 5 μM ZA for 2 days. Human Vγ9Vδ2 T cells were MACS purified (day 14)and co-cultured with the cancer cells for 24 h. Cytotoxicity of Vγ9Vδ2 Tcells towards non-treated and ZA-treated target cells was determined bymeasuring remaining luciferase activity or calcein fluorescence; cf.FIG. 11b . Target cells (NUGC-4 and CLS103) were pretreated with or w/o5 μM ZA for 2 days and subsequently incubated for 4 h with mitomycin c(50 MI) to stop proliferation. MACS purified human 14 d old restingVγ9Vδ2 T cells and ³H-thymidine were added to the target cells andco-cultures were incubated for 48 h at 37° C. Proliferation wasdetermined by measuring ³H thymidine incorporation in the DNA using aMicroBeta scintillation counter. Proliferation of target cells nottreated with ZA and w/o Vγ9Vδ2 T cells was set to 100%; cf. FIG. 11 c.

As shown in FIGS. 11b and 11c the ZA-pulsed human cancer cells activatedVγ9Vδ2 T cells in terms of cytotoxicity (5-10 fold) and proliferation(1.4-1.8 fold), whereas the murine cancer cell line CLS103 failed toelicit these effects on Vγ9Vδ2 T cells.

Example 9: ZA/IL-2 Treatment Affects Composition of PBMC Cultures

Growth and differentiation of specific cell types in PBMC culturesdepends on presence of cytokines. These components are either added tothe medium (e.g. growth factors present in the serum, IL-2) or secretedby the immune cells themselves. Which type of cells evolves also dependson the initial composition of the PBMCs and on genetic endowments. Toanalyze the overall increase in effector cells (NK cells and Vγ9Vδ2 Tcells) PBMCs of 10 different donors were grown in the presence of 300U/ml IL-2 and with or w/o 1 μM ZA for 14 days. The amount of effectorcells within the lymphocyte population was identified by multicolor FACSstaining using CD3, CD16, CD56, Vγ9 and Vδ2 antibodies. CD3−CD56+CD16+cells represent NK cells and CD3+Vγ9+Vδ2+ represent Vγ9Vδ2 T cells.

Multicolor FACS analysis revealed that upon IL-2 treatment mainly NKcells develop, whereas in ZA/IL-2 treated cultures Vγ9Vδ2 T cells arepredominantly expanded (FIG. 12).

Example 10: ZA/IL-2 Treatment Generates Vγ9Vδ2+ Effector Memory T Cells

Subpopulations of T lymphocytes can be delineated with the help of twosurface markers, the high m.w. isoform of the common lymphocyte antigenCD45RA and the chemokine receptor CCR7. CCR7+ naive and central-memory(CM) T cells are characterized by the ability to repeatedly circulateinto lymph nodes and encounter antigen. In contrast, effector-memory(EM) and effector T lymphocytes RA+ (TEMRA) down-regulate CCR7 andappear specialized in migrating to peripheral nonlymphoid tissues e.g.to infected or tumor sites. The EM cells can be further subdivided basedon differential CD27 and CD28 expression.

Progressive loss of CD28 and CD27 surface expression is concomitant withup-regulation of cytolytic capacity of the cells. In addition the levelof CD57 correlates with the expression of granzymes and perforins andthus represents a third marker displaying cytotoxicity/cell maturation.

PBMCs were cultivated with or w/o 1 μM ZA and 300 U/ml IL-2 for 14 days.The expression of the different surface markers was determined bymulticolor FACS analysis on day 0 (PBMCs) and day 14. Naive cells areCD45RA+ CCR7+, central memory cells (CM) are CD45RA− CCR7+, TEMRA areCD45RA+ CCR7- and effector memory cells (EM) are negative for bothmarkers; cf. FIG. 13a . Furthermore, cytolytic activity of the Vγ9Vδ2T-cells was determined by staining for CD27 and CD57 markers; cf. FIG.13b,c . In addition, the development of NK cell-like characteristicsimportant for ADCC activity was analyzed by staining CD3+ cells withCD16 (antibody binding) and CD56 (adhesion); cf. FIG. 13 d.

Multicolor FACS analysis of the Vγ9Vδ2 T cells revealed that ZA/IL-2treatment clearly stimulated development of Vγ9Vδ2 T cells of the EMtype which are CD27- and CD57+(FIG. 13b-c ). In addition to enhancedcytolytic activity, an increase in the level of CD16 and CD56, which areknown from NK cells (CD3−CD16+CD56+) to be involved in ADCC was observedin the CD3+ population (FIG. 13d ).

Taken together, these data imply that ZA treatment of PBMCs results inthe development of CD16+Vγ9+Vδ2+ effector memory T cells, which are ableto migrate to peripheral nonlymphoid tissues and which display markersof high cytolytic activity. In combination with the IMAB362 tumortargeting antibody these cells are extremely well harnessed to migrateto, target and kill tumor cells.

Example 11: ZA/IL-2 Expanded Vγ9Vδ2 T Cells are Potent Effectors forIMAB362-Mediated CLDN18.2 Dependent ADCC

Similar to NK cells, the ZA/IL-2 expanded Vγ9Vδ2 T cells are positivefor CD16 (see FIGS. 9 and 13), the FcγRIII receptor via which acell-bound antibody triggers ADCC. To evaluate whether Vγ9Vδ2 T cellsare capable of inducing potent ADCC in conjunction with IMAB362 a seriesof experiments has been performed.

PBMCs derived from 2 different donors (#1 and #2) were cultivated inmedium with 300 U/ml IL-2 and with or w/o 1 μM ZA. After 14 days cellswere harvested and added with increasing concentrations (0.26 ng/ml-200μg/ml) of IMAB362 to NUGC-4 cells expressing CLDN18.2. Specific killingwas determined in luciferase assays; cf. FIG. 14a . FIG. 14b,c gives anoverview of ADCC assays performed with 27 donors grown in 300 U/ml IL-2and either with or w/o ZA. NUGC-4 served as target cells. For eachdonor, the EC₅₀ values (b) calculated from the dose-response curves andthe maximum specific killing rate at a dose of 200 μg/ml IMAB362 (c)were scored in the scatter plots.

Strong IMAB362-dependent ADCC activity was observed againstCLDN18.2-positive NUGC-4 cells using PBMCs cultivated for 14 days withZA/IL-2 (FIG. 14a ). Using ZA/IL-2-treated PBMC cultures, ADCC dependson the presence of Vγ9Vδ2 T cells (FIGS. 12 and 15). If cells arecultured without ZA, ADCC activity is reduced for most donors. In thesecultures, residual ADCC activity is NK-cell dependent (FIGS. 11 and 14).By testing more than 20 donors, ADCC assays reveal that ZA/IL-2treatment of PBMCs improves the EC₅₀ and maximum specific killing ratesas compared to PBMCs cultured with IL-2 alone.

Furthermore, PBMCs of two different donors (#1+#2) were cultured with 1μM ZA and 300 U/ml IL-2. These effector cell cultures were used in ADCCassays with CLDN18.2-positive (NUGC-4, KATO III) and negative (SK-BR-3)human target cell lines (E:T ratio 40:1). Increasing amounts (0.26ng/ml-200 μg/ml) of IMAB362 antibody were added. ADCC was measured inluciferase assays; cf. FIG. 15a . Same experiment as described in (a)was performed with NUGC-4 target cells and effector cells harvested fromcultures treated with ZA/IL-2 at different time points; cf. FIG. 15b .Same experiment as described in (a) was performed using NUGC-4 as targetcells; cf. FIG. 15c . The ZA/IL-2 expanded cells were either useddirectly, or Vγ9Vδ2 T cells were purified from the cultures using TCRγδMACS sorting (Miltenyi Biotech). A purity of more than 97.0% Vγ9Vδ2 Tcells in lymphocytes was obtained.

Strong ADCC activity against CLDN18.2-positive, but notCLDN18.2-negative human tumor cell lines has been observed (FIG. 15a ).Furthermore, no ADCC activity is obtained with isotype controlantibodies (not shown). In the course of ZA/IL-2 treatment ADCC lyticactivity increases over time for a fraction of donors (FIG. 15b ). Thedose/effect curve of IMAB362 shifts upward and to the left showingimproved EC₅₀ values and maximum lysis rates over time. Compared tounconditioned PBMC, the Vγ9Vδ2 effector T cells enriched by ZA/IL-2treatment are capable of reaching a higher maximum killing rate ofCLDN18.2-positive target cells plus they require lower concentrations ofIMAB362 for the same killing rate.

To confirm that Vγ9Vδ2 T cells are the reservoir for lytic activity,these cells were isolated with >97% purity by magnetic cell sorting fromZA/IL-2-cultured PBMC populations on day 14. The ADCC activity inconjunction with IMAB362 is retained and partly improved due to higherpurity. These data confirm that Vγ9Vδ2 T cells are mainly responsiblefor the ADCC activity observed with 14 days old PBMC cultures (FIG. 15c).

Example 12: Treatment of Target Cell Lines with ZA/IL-2 does not AffectSurface Expression of CLDN18.2

IMAB362 triggered modes of action are strictly dependent on the presenceand amount of extracellular detectable CLDN18.2. Therefore the influenceof ZA/IL-2 treatment on CLDN18.2 surface density has been analyzed byflow cytometry using endogenous CLDN18.2 expressing NUGC-4 and KATO IIIcell lines. Specifically, flow cytometric analysis of IMAB362 binding onunpermeabilized NUGC-4 gastric cancer cells pretreated with ZA/IL-2 orZA/IL-2+EOF or ZA/IL-2+5-FU/OX for 72 hours was performed. ZA/IL-2treatment in vitro reveals no change in amount of CLDN18.2 surfacelocalization; cf. FIG. 16.

Example 13: Augmentation of IMAB362-Mediated ADCC by ZA/IL-2 Treatmentof PBMCs is not Compromised by EOF Pretreatment

Chemotherapeutic agents compromise cell proliferation. In contrastZA/IL-2 treatment triggers expansion of Vγ9Vδ2 T cells. To analyze theinfluences of these opposing interactions on effector cells, PBMCs of 6healthy donors were cultured with ZA/IL-2 or ZA/IL-2+EOF for 8 daysbefore application in ADCC assays (E:T ratio 15:1). IMAB362concentrations resulting in 50% ADCC mediated lysis of untreated NUGC-4target cells (EC₅₀) were determined.

Augmentation of IMAB362 induced ADCC of NUGC-4 cells due to PBMCtreatment with ZA/IL-2 is not significantly altered by combinedtreatment of PBMCs with EOF (FIG. 17).

Example 14: In Vivo Targeting of IMAB362 to CLDN18.2-Positive Tumors andAntitumoral Effects of IMAB362 on Human Tumor Cell Xenografts in NudeMice

To investigate in vivo tumor cell targeting of IMAB362, 80 μg Dyelight®680-labeled antibody was administered intravenously to nude mice thatwere xenografted subcutaneously with the human gastric cancer cell lineNUGC-4. NUGC-4 cells display surface expression of CLDN18.2 as well asof HER2/neu (target of trastuzumab), but are negative for CD20. Controlstudies were conducted injecting NUGC-4 engrafted groups of mice witheither Dyelight 680-labeled trastuzumab (positive control group) orDyelight® 680-labeled rituximab (negative control). IMAB362 accumulatesstrongly and exclusively in the tumor xenografts, as demonstrated bylive imaging of mice using a Xenogen® fluorescence imaging system 24hours after i.v. injection of antibodies (FIG. 18). IMAB362 isefficiently retained in the target-positive tumor and detectable incomparable intensity even after 120 hours (FIG. 18). Trastuzumab is alsodetected exclusively in the xenografts 24 hours after injection. Thetrastuzumab signal is rapidly washed out within 120 hours afterinjection. No signal is detected with rituximab.

Furthermore, IMAB362 was used to treat nude mice bearingCLDN18.2-positive xenograft tumors. Early treatment model studies (withadministrations of IMAB362 as early as 3 days after tumor cellsinoculation) were conducted. Moreover, advanced tumor treatmentexperiments were initiated up to 9 days after tumor cell inoculationwhen tumors had reached volumes of about 60-120 mm³.

Nude mice were subcutaneously inoculated with 1×10⁷ HEK293˜CLDN18.2transfectants. Treatment of 10 mice per group started 3 days after tumorinoculation. Mice were treated with 200 μg IMAB362, infliximab asisotype control and PBS twice per week for 6 weeks alternatingintravenous and intraperitoneal routes of application. Whereas all micein the groups treated with either PBS or isotype control died within70-80 days, animals treated with IMAB362 had a survival benefit (FIG.19). Not only time to death was prolonged, but 4 of 10 mice survived theentire observation period of 210 days.

Treatment of 9 to 10 mice per group was initiated when mean tumorvolumes reached 88 mm³ (62-126 mm³). Prior to treatment mice werestratified into test groups to ensure comparable tumor sizes in allgroups. Mice were treated with 200 μg IMAB362, isotype control or PBStwice per week for 6 weeks alternating intravenous and intraperitonealroutes of application. All mice in the groups treated with either PBS orisotype control died within 50-100 days. Animals treated with IMAB362had a survival benefit, with nearly a doubling of median survival time(47 versus 25 days). Three of these mice survived the entire observationperiod (FIG. 20). Importantly, antitumoral efficacy in vivo depends onthe presence of the target on the tumor cells. No antitumoral effects ofIMAB362 treatment were seen in mice engrafted with CLDN18.2-negativeHEK293 tumor cells.

The NUGC-4 gastric tumor model was used to investigate the efficacy ofIMAB362 against cancer cells with endogenous expression of CLDN18.2.NUGC-4 cells grow aggressively in nude mice.

1×10⁷ NUGC-4 gastric cancer cells were injected subcutaneous into theleft flank of athymic nude mice (n=9 for IMAB362 group; n=8 for controlgroups). IMAB362 (200 μg per injection) and controls were applied twiceweekly alternating i.v. and i.p., starting 6 days after tumorinoculation with i.v. injection. Tumor sizes were monitored twiceweekly. Data presented in FIG. 21a are means with SEM. Tumor growth ofmice treated with IMAB362 was significant inhibited compared to micetreated with controls (*p<0.05). FIG. 21b shows tumor volumes at day 21after tumor inoculation. Tumor volumes of IMAB362 treated mice weresignificant smaller than tumors of control mice (*p<0.05).

When 1×10⁷ tumor cells are inoculated in mice, the median survival timeof untreated mice is not longer than 25 days. Treatment with IMAB362,cetuximab, trastuzumab or isotype and buffer controls was initiated whentumor volumes reached a mean size of about 109 mm³ (63-135 mm³). Micewere stratified size-dependently into treatment groups (FIG. 21).IMAB362 was shown to significantly reduce tumor growth rate. Nosignificant reduction of tumor growth as compared to saline or antibodycontrols was observed for this aggressively growing tumor model. Thedelay in tumor growth was associated with a non-significantly increasedmedian survival time of IMAB362 treated mice (31 days versus 25 days).

Antitumor activity of IMAB362 was examined with two human gastriccarcinoma xenograft models using NCI-N87 or NUGC-4 cells with lentiviraltransduction of IMAB362 target CLDN18.2 (NCI-N87˜CLDN18.2 andNUGC-4˜CLDN18.2).

NCI-N87˜CLDN18.2 xenograft tumors were inoculated subcutaneously byinjection of 1×10⁷ NCI-N87˜CLDN18.2 cells into the flank of 8 nude mice(female, 6 weeks old) per treatment group. Treatment started 5 daysafter tumor inoculation by intravenous injection of 800 μg IMAB362 orwith 200 μl 0.9% NaCl for saline control group. Intravenousadministration was continued weekly for the whole observation time.Tumor size and animal health was monitored semi-weekly. FIG. 22a showsthe effects of IMAB362 treatment on tumor growth. The size of s.c.tumors was measured twice weekly (mean+SEM, ***p<0.001). FIG. 22b showsKaplan-Meier survival plots. Mice were sacrificed, when tumor reached avolume of 1400 mm³.

Thus, continuous IMAB362 treatment inhibited highly significant(p<0.001) tumor growth of NCI-N87˜CLDN18.2 gastric carcinoma xenografts(FIG. 22a ). The delay in tumor growth was associated with asignificantly (p<0.05) longer survival time of IMAB362 treated mice(FIG. 22b ).

IMAB362 immunotherapy of rapid growing NUGC-4˜CLDN18.2 xenograftsresulted in significant (p<0.05) smaller tumor sizes at day 14 oftreatment. After the first two weeks of IMAB362 treatment tumorprogession of NUGC-4˜CLDN18.2 was very aggressive. However, theinhibition of NUGC-4˜CLDN18.2 tumor growth until day 14 of treatmentresulted in significantly (p<0.05) longer survival of IMAB362 treatedmice.

In summary, IMAB362 was highly effective in treatment of gastriccarcinoma xenografts showing significant retardation of tumorprogression and prolonged survival in endogenous CLDN18.2-positive tumormodels. In very aggressive tumor model systems these antitumoral effectsof IMAB362 are less prominent but nonetheless significant, emphasizingthe strong antitumoral capacity of IMAB362.

Example 15: Antitumoral Effects of IMAB362 Combined with Chemotherapy inMouse Tumor Models

In vitro, IMAB362-mediated ADCC is more efficient on human gastriccancer cells pretreated with combinations of chemotherapeutic agentsincluding the EOF and 5-FU+OX. Therefore, antitumoral impact ofcombining these compounds with IMAB362 was investigated in vivo in mousetumor models.

NCI-N87˜CLDN18.2 xenograft tumors were inoculated by injection of 1×10⁷NCI-N87˜CLDN18.2 cells subcutaneous into the flank of 9 mice for eachtreatment group. Tumor bearing mice were treated according to EOFregimen with 1.25 mg/kg epirubicin, 3.25 mg/kg oxaliplatin and 56.25mg/kg 5-fluorouracil intraperitoneal on day 4, 11, 18 and 25 after tumorinoculation, followed by intravenous injection of 800 μg IMAB362 24hours after chemotherapy administration. IMAB362 treatment was continuedweekly. Tumor size and animal health was monitored semi-weekly. FIG. 23ashows the effects of combined treatment on tumor growth. The size ofs.c. tumors was measured twice weekly (mean+SEM; *p<0.05). FIG. 23bshows Kaplan-Meier survival plots. Mice were sacrificed, when tumorreached a volume of 1400 mm³.

NCI-N87˜CLDN18.2 tumor bearing nude mice treated with IMAB362 or EOFregimen showed highly significant suppressed tumor growth compared tocontrol mice. Additional IMAB362 treatment in combination with EOFchemotherapy resulted in significantly (p<0.05) higher tumor growthinhibition than treatment with EOF regimen alone (FIG. 23a ). Mediansurvival of mice in saline control group was 59 days. Weekly IMAB362treatment of mice prolonged significantly median survival to 76 dayssimilar to survival of mice in EOF group with a median survival of 76days, too. But combined treatment with IMAB362 and EOF augmented mediansurvival to 81 days (FIG. 23b ).

Xenograft tumors were inoculated by injection of 1×10⁷ NUGC-4˜CLDN18.2cells subcutaneous into the flank of 10 nude mice (female, six weeksold) per treatment group. Mice were treated on day 3, 10, 17 and 24 withchemotherapeutic agents. IMAB362 treatment was continued weekly. FIG.24a shows tumor growth curves of s.c. NUGC-4˜CLDN18.2 xenografts(mean+SEM). FIG. 24b shows Kaplan-Meier survival plots (Log-rank(Mantel-Cox) Test, **p<0.01).

Subcutaneous NUGC-4˜CLDN18.2 xenograft tumors grow very aggressively.Nevertheless IMAB362 treatment of tumor bearing nude mice inhibitedsignificantly the tumor growth compared to saline treated control group.In combined therapy with EOF, IMAB362 effects on NUGC-4˜CLDN18.2 tumorgrowth was masked by growth inhibition due to EOF treatment, showing noincreased tumor growth inhibition compared to treatment with EOF alone(FIG. 24a ). However, median survival of mice treated with IMAB362 andEOF regimen was highly significant (p<0.01) prolonged, compared tosurvival of mice treated with EOF alone (FIG. 24b ).

Example 16: ZA/IL-2 Expanded Vγ9Vδ2 T Cells Improve IMAB362-MediatedControl of Advanced Tumors In Vivo

To investigate combined activity of IMAB362 and ZA/IL-2 generated γδ Tcells in mouse systems, we resorted to NSG mice. NSG mice lack mature Tcells, B cells, natural killer (NK) cells, multiple cytokine signalingpathways, and they have many defects in innate immunity, whereas theniches in the primary and secondary immunological tissues are permissiveto colonization by human immune cells.

NSG mice were inoculated subcutaneously with 1×10⁷ CLDN18.2-transfectedHEK293 cells. On the same day, mice received 8×10⁶ human PBMCs enrichedfor Vγ9Vδ2 T cells, which were cultured for 14 days in ZA-supplementedmedium. Moreover, mice were injected with 50 μg/kg ZA and 5000 U IL-2(Proleukin). To maintain human T cells functional, IL-2 was administeredsemi-weekly and ZA weekly. When HEK293˜CLDN18.2 tumors becamemacroscopically visible, semi-weekly treatment with 200 μg of IMAB362was started. In addition to 9 mice treated as described, two controlgroups of mice were established. One group did not receive human γδ Tcells, the other group was treated with an isotype control antibodyinstead of IMAB362. Outgrowth of CLDN18.2-positive tumors in micetreated with IMAB362 in the presence of human γδ T cells and ZA wassignificantly inhibited and nearly abrogated, whereas in mice eithertreated with an isotype control antibody or lacking human T celleffectors, tumors grew aggressively and mice had to be terminatedprematurely (FIG. 25).

Example 17: Antitumoral Effects of IMAB362 Combined with Chemotherapy inMouse Tumor Models

Antitumor activity of IMAB362 in combination with chemotherapy wasexamined in subcutaneous gastric carcinoma allografts in immunocompetentoutbred NMRI mice using CLS-103 cells with lentiviral transduction ofmurine cldn18.2 (CLS-103˜cldn18.2).

CLS-103˜cldn18.2 allograft tumors were inoculated by injection of 1×10⁶CLS-103˜cldn18.2 cells subcutaneous into the flank of 10 NMRI mice foreach treatment group. Tumor bearing mice were treated with 1.25 mg/kgepirubicin, 3.25 mg/kg oxaliplatin and 56.25 mg/kg 5-fluorouracil (EOF)intraperitoneal on day 3, 10, 17 and 24 after tumor inoculation,followed by intravenous injection of 800 μg IMAB362 24 hours after eachchemotherapy administration. IL-2 was administered semi-weekly bysubcutaneous injection of 3000 IE. After end of chemotherapy, IMAB362and IL-2 treatment was continued for the whole observation period. Tumorsize and animal health were monitored semi-weekly. Mice were sacrificed,when tumor reached a volume of 1400 mm³ or tumors became ulcerous.

As can be seen in FIG. 26, CLS-103˜cldn18.2 tumor bearing NMRI micetreated with IMAB362 or EOF alone showed no significant tumor growthinhibition compared to saline control group. In contrast, thecombination of EOF chemotherapy and IMAB362 treatment resulted insignificantly higher tumor growth inhibition and in prolonged survivalof tumor bearing mice. These observations indicate the existence ofadditive or even synergistic therapeutic effects by combination of EOFchemotherapy and IMAB362 immunotherapy. IL-2 treatment showed no effecton tumor growth.

1-36. (canceled)
 37. A method of treating a cancer disease characterizedby cells expressing claudin 18 splice variant 2, comprisingadministering to a patient an antibody having the ability of binding toclaudin 18 splice variant 2 (CLDN18.2) in combination with an agentstabilizing or increasing expression of CLDN18.2, wherein the agentstabilizing or increasing expression of CLDN18.2 is selected from thegroup consisting of anthracyclines, platinum compounds, nucleosideanalogs, taxanes, camptothecin analogs, prodrugs thereof, andcombinations thereof, wherein the antibody comprises a heavy chainvariable region (VH) comprising a set of complementarity-determiningregions CDR1, CDR2 and CDR3 and a light chain variable region (VL)comprising a set of complementarity-determining regions CDR1, CDR2 andCDR3, wherein said VH and VL is selected from the group consisting of:(i) VH: CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions 70-77 ofSEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO: 14, VL: CDR1:positions 49-53 of SEQ ID NO: 21, CDR2: positions 71-73 of SEQ ID NO:21, CDR3: positions 110-118 of SEQ ID NO: 21, (ii) VH: CDR1: positions45-52 of SEQ ID NO: 15, CDR2: positions 70-77 of SEQ ID NO: 15, CDR3:positions 116-126 of SEQ ID NO: 15, VL: CDR1: positions 47-58 of SEQ IDNO: 20, CDR2: positions 76-78 of SEQ ID NO: 20, CDR3: positions 115-123of SEQ ID NO: 20, (iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16,CDR2: positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQID NO: 16, VL: CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO: 22, (iv)VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions 69-76 of SEQID NO: 18, CDR3: positions 115-125 of SEQ ID NO: 18, VL: CDR1: positions47-58 of SEQ ID NO: 25, CDR2: positions 76-78 of SEQ ID NO: 25, CDR3:positions 115-122 of SEQ ID NO: 25, (v) VH: CDR1: positions 45-53 of SEQID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 23,CDR2: positions 76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQID NO: 23, (vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:19, VL: CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions 76-78 ofSEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO: 26, (vii) VH: CDR1:positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO:19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58of SEQ ID NO: 27, CDR2: positions 76-78 of SEQ ID NO: 27, CDR3:positions 115-123 of SEQ ID NO: 27, and (viii) VH: CDR1: positions 45-53of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3:positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-52 of SEQ IDNO: 28, CDR2: positions 70-72 of SEQ ID NO: 28, CDR3: positions 109-117of SEQ ID NO:
 28. 38. The method of claim 37, comprising administeringan agent stimulating γδ T cells, wherein the agent stimulating γδ Tcells is a bisphosphonate.
 39. The method of claim 37, wherein the agentstabilizing or increasing expression of CLDN18.2 comprises at least oneanthracycline, at least one platinum compound and at least one of5-fluorouracil and prodrugs thereof.
 40. The method of claim 37, whereinthe taxane is selected from docetaxel and paclitaxel.
 41. The method ofclaim 37, wherein the camptothecin analog is selected from the groupconsisting of irinotecan and topotecan.
 42. The method of claim 37,wherein the agent stabilizing or increasing expression of CLDN18.2comprises (i) epirubicin, oxaliplatin and 5-fluorouracil, (ii)epirubicin, oxaliplatin and capecitabine, (iii) epirubicin, cisplatinand 5-fluorouracil, (iv) epirubicin, cisplatin and capecitabine, or (v)folinic acid, oxaliplatin and 5-fluorouracil.
 43. The method of claim38, wherein the bisphosponate is selected from the group consisting ofzoledronic acid, clodronic acid, ibandronic acid, pamidronic acid,risedronic acid, minodronic acid, olpadronic acid, alendronic acid,incadronic acid and salts thereof.
 44. The method of claim 37,comprising administering to the patient interleukin-2.
 45. The method ofclaim 38, comprising administering to the patient interleukin-2.
 46. Themethod of claim 37, wherein the antibody having the ability of bindingto CLDN18.2 mediates cell killing by one or more of complement dependentcytotoxicity (CDC) mediated lysis, antibody dependent cellularcytotoxicity (ADCC) mediated lysis, induction of apoptosis andinhibition of proliferation.
 47. The method of claim 37, wherein theantibody having the ability of binding to CLDN18.2 is an antibodyselected from the group consisting of (i) an antibody produced by and/orobtainable from a clone deposited under the accession no. DSM ACC2737,DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSMACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSMACC2808, or DSM ACC2809, (ii) an antibody which is a chimerized orhumanized form of the antibody under (i), and (iii) an antibodycomprising the antigen binding portion or antigen binding site, inparticular the variable region, of the antibody under (i) and preferablyhaving the specificity of the antibody under (i).
 48. The method ofclaim 37, wherein the cancer disease is selected from the groupconsisting of esophageal adenocarcinoma, pancreatic adenocarcinoma, lungadenocarcinoma, cancer of the stomach, cancer of the esophagus, inparticular the lower esophagus, gastric cancer, cancer of theeso-gastric junction and gastroesophageal cancer.
 49. The method ofclaim 39, wherein the at least one anthracycline is epirubicin.
 50. Themethod of claim 39, wherein the at least one platinum compound isselected from the group consisting of oxaliplatin and cisplatin.
 51. Themethod of claim 39, wherein the at least one anthracycline isepirubicin, the at least one platinum compound is oxaliplatin, and theat least one of 5-fluorouracil and prodrugs thereof is 5-fluorouracil orcapecitabine.
 52. The method of claim 37, wherein the antibody has aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 29, 30, 31, 33, and
 34. 53. Themethod of claim 37, wherein the antibody has a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 35, 36, 37, 38, 40, 41, 42,
 43. 54. The methodof claim 37, wherein the antibody comprises a combination of heavy chainvariable region (VH) and light chain variable region (VL) selected fromthe group consisting of: (i) the VH comprises an amino acid sequencerepresented by SEQ ID NO: 29 and the VL comprises an amino acid sequencerepresented by SEQ ID NO: 36, (ii) the VH comprises an amino acidsequence represented by SEQ ID NO: 30 and the VL comprises an amino acidsequence represented by SEQ ID NO: 35, (iii) the VH comprises an aminoacid sequence represented by SEQ ID NO: 31 and the VL comprises an aminoacid sequence represented by SEQ ID NO: 37, (iv) the VH comprises anamino acid sequence represented by SEQ ID NO: 33 and the VL comprises anamino acid sequence represented by SEQ ID NO: 40, (v) the VH comprisesan amino acid sequence represented by SEQ ID NO: 34 and the VL comprisesan amino acid sequence represented by SEQ ID NO: 38, (vi) the VHcomprises an amino acid sequence represented by SEQ ID NO: 34 and the VLcomprises an amino acid sequence represented by SEQ ID NO: 41, (vii) theVH comprises an amino acid sequence represented by SEQ ID NO: 34 and theVL comprises an amino acid sequence represented by SEQ ID NO: 42, and(viii) the VH comprises an amino acid sequence represented by SEQ ID NO:34 and the VL comprises an amino acid sequence represented by SEQ ID NO:43.
 55. A method of treating a cancer disease characterized by cellsexpressing claudin 18 splice variant 2, comprising administering to apatient an antibody having the ability of binding to claudin 18 splicevariant 2 (CLDN18.2) in combination with an agent stabilizing orincreasing expression of CLDN18.2, wherein the agent stabilizing orincreasing expression of CLDN18.2 comprises at least one platinumcompound and at least one of 5-fluorouracil and prodrugs thereof,wherein the antibody comprises a heavy chain variable region (VH)comprising a set of complementarity-determining regions CDR1, CDR2 andCDR3 and a light chain variable region (VL) comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3, wherein said VHand VL is selected from the group consisting of: (i) VH: CDR1: positions45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3:positions 116-125 of SEQ ID NO: 14, VL: CDR1: positions 49-53 of SEQ IDNO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118of SEQ ID NO: 21, (ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2:positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO:15, VL: CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 ofSEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20, (iii) VH: CDR1:positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO:16, CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1: positions 47-52of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3:positions 109-117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44-51 ofSEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions115-125 of SEQ ID NO: 18, VL: CDR1: positions 47-58 of SEQ ID NO: 25,CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQID NO: 25, (v) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:19, VL: CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 ofSEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23, (vi) VH: CDR1:positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO:19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3:positions 115-123 of SEQ ID NO: 26, (vii) VH: CDR1: positions 45-53 ofSEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 27,CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQID NO: 27, and (viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:19, VL: CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 ofSEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO:
 28. 56. The methodof claim 37, wherein the agent stabilizing or increasing expression ofCLDN18.2 comprises (i) oxaliplatin and 5-fluorouracil, (ii) oxaliplatinand capecitabine, (iii) cisplatin and 5-fluorouracil, (iv) cisplatin andcapecitabine, or (v) folinic acid, oxaliplatin, and 5-fluorouracil. 57.The method of claim 55, wherein the agent stabilizing or increasingexpression of CLDN18.2 comprises (i) oxaliplatin and 5-fluorouracil,(ii) oxaliplatin and capecitabine, (iii) cisplatin and 5-fluorouracil,(iv) cisplatin and capecitabine, or (v) folinic acid, oxaliplatin, and5-fluorouracil.
 58. The method of claim 55, wherein the agentstabilizing or increasing expression of CLDN18.2 comprises folinic acid,oxaliplatin, and 5-fluorouracil.
 59. The method of claim 55, wherein theantibody having the ability of binding to CLDN18.2 mediates cell killingby one or more of complement dependent cytotoxicity (CDC) mediatedlysis, antibody dependent cellular cytotoxicity (ADCC) mediated lysis,induction of apoptosis and inhibition of proliferation.
 60. The methodof claim 55, wherein the antibody having the ability of binding toCLDN18.2 is an antibody selected from the group consisting of (i) anantibody produced by and/or obtainable from a clone deposited under theaccession no. DSM ACC2810, (ii) an antibody which is a chimerized orhumanized form of the antibody under (i), and (iii) an antibodycomprising the antigen binding portion or antigen binding site, inparticular the variable region, of the antibody under (i) and preferablyhaving the specificity of the antibody under (i).
 61. The method ofclaim 55, wherein the cancer disease is selected from the groupconsisting of esophageal adenocarcinoma, pancreatic adenocarcinoma, lungadenocarcinoma, cancer of the stomach, cancer of the esophagus, inparticular the lower esophagus, gastric cancer, cancer of theeso-gastric junction and gastroesophageal cancer.
 62. The method ofclaim 55, wherein the antibody has a heavy chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 29, 30, 31, 33, and
 34. 63. The method of claim 55, whereinthe antibody has a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 35, 36, 37,38, 40, 41, 42,
 43. 64. The method of claim 55, wherein the antibodycomprises a heavy chain variable region (VH) comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 and a lightchain variable region (VL) comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3, wherein said VHand VL is selected from the group consisting of: (i) VH: CDR1: positions45-52 of SEQ ID NO: 14, CDR2: positions 70-77 of SEQ ID NO: 14, CDR3:positions 116-125 of SEQ ID NO: 14, VL: CDR1: positions 49-53 of SEQ IDNO: 21, CDR2: positions 71-73 of SEQ ID NO: 21, CDR3: positions 110-118of SEQ ID NO: 21, (ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2:positions 70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO:15, VL: CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions 76-78 ofSEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO: 20, (iii) VH: CDR1:positions 45-52 of SEQ ID NO: 16, CDR2: positions 70-77 of SEQ ID NO:16, CDR3: positions 116-124 of SEQ ID NO: 16, VL: CDR1: positions 47-52of SEQ ID NO: 22, CDR2: positions 70-72 of SEQ ID NO: 22, CDR3:positions 109-117 of SEQ ID NO: 22, (iv) VH: CDR1: positions 44-51 ofSEQ ID NO: 18, CDR2: positions 69-76 of SEQ ID NO: 18, CDR3: positions115-125 of SEQ ID NO: 18, VL: CDR1: positions 47-58 of SEQ ID NO: 25,CDR2: positions 76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQID NO: 25, (v) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:19, VL: CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions 76-78 ofSEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO: 23, (vi) VH: CDR1:positions 45-53 of SEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO:19, CDR3: positions 117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58of SEQ ID NO: 26, CDR2: positions 76-78 of SEQ ID NO: 26, CDR3:positions 115-123 of SEQ ID NO: 26, (vii) VH: CDR1: positions 45-53 ofSEQ ID NO: 19, CDR2: positions 71-78 of SEQ ID NO: 19, CDR3: positions117-128 of SEQ ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 27,CDR2: positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQID NO: 27, and (viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:19, VL: CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions 70-72 ofSEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO:
 28. 65. The methodof claim 64, wherein the agent stabilizing or increasing expression ofCLDN18.2 comprises folinic acid, oxaliplatin, and 5-fluorouracil. 66.The method of claim 64, wherein the antibody is a chimeric antibodycomprising a human kappa light chain constant region and a human IgG1heavy chain constant region.
 67. The method of claim 64, wherein thehuman kappa light chain constant region is allotype Km(3) and/or thehuman IgG1 heavy chain constant region is allotype G1m(3).
 68. Themethod of claim 64, wherein the human kappa light chain constant regioncomprises an amino acid sequence represented by SEQ ID NO: 12 and thehuman IgG1 heavy chain constant region comprises an amino acid sequencerepresented by SEQ ID NO:
 13. 69. A method of treating a cancer diseasecharacterized by cells expressing claudin 18 splice variant 2,comprising administering to a patient an antibody having the ability ofbinding to claudin 18 splice variant 2 (CLDN18.2) in combination with anagent stabilizing or increasing expression of CLDN18.2, wherein theagent stabilizing or increasing expression of CLDN18.2 comprises (i)capecitabine and oxaliplatin), (ii) folinic acid, 5-fluorouracil andoxaliplatin, or (iii) epirubicin, oxaliplatin and 5-fluorouracil,including prodrugs thereof, prodrugs thereof, and combinations thereof,wherein the antibody comprises a heavy chain variable region (VH) havinga CDR1 of positions 45-52 of SEQ ID NO: 17, a CDR2 of positions 70-77 ofSEQ ID NO: 17, and a CDR3 of positions 116-126 of SEQ ID NO: 17, and alight chain variable region (VL) having a CDR1 of positions 47-58 of SEQID NO: 24, a CDR2 of positions 76-78 of SEQ ID NO: 24, and a CDR3 ofpositions 115-123 of SEQ ID NO: 24.